Biological and biochemical aspects of microbial growth on C1 compounds.

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BIOLOGICAL AND

.1764

BIOCHEMICAL ASPECTS OF MICROBIAL GROWTH ON Cl COMPOUNDS John Colby, Howard Dalton 1, and Roger Whittenbury Department of Biological Sciences, University of Warwick, Coventry, England

CONTENTS INTRODUCfION ......................................................................................................... . THE TAXONOMY AND BIOLOGY OF METHANE-OXIDIZING MICROBES Taxonomy and Nomenclature................................................................................... . Intracytoplasmic Membranes. .................................................................................... . Phospholipid and Fatty Acid Content ....................................................................... . Tricarboxylic Acid Cycle ........................................................................................... . Carbon Assimilation Pathways ................................................................................. . The Relationship 0/ Methylotrophy to Autotrophy ................................................... . MICROBIAL ASSIMILATION OF C, COMPOUNDS ............................................ Ribulose Diphosphate Pathway 0/ CO2 Assimilation ............................................... . RMP Pathway ............................................................................................................ Do Methanol-Utilizing Yeasts Use a Modified RMP Pathway? ............................... The Serine Pathway................................................................................................... . Genetic Regulation 0/ C] Metabolism ....................................................................... .

MICROBIAL OXIDATION OF C, COMPOUNDS .................................................. Oxidation 0/ Carbon Monoxide ............................................................................... . Oxidation 0/ Methane and Methanol ........................................................................ Oxidation 0/ Methyl Amines ..................................................................................... . Oxidation 0/ Formaldehyde....................................................................................... .

ENZYMOLOGY OF C, OXIDATIONS ...................................................................... Methane Monooxygenase............................................................................................ Methanol Dehydrogenase (EC 1.1.99.8) .................................................................... Methylamine Dehydrogenase..............................•........•.............................................. Trimethylamine Dehydrogenase (EC 1.5.99. 7)..........................................................

482 482 482 484 485 485 485 487 488 489 490 493 494 497 497 498 500 501 506 507 507 510 511 512

'Requests for reprints should be addressed to Dr. Dalton.

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0066-4227/79/ 1001-048 1$01.00

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COLBY, DALTON & WHITTENBURY


INTRODUCTION

CI-utilizing microorganisms are recognized by their ability to use, as sole carbon source for growth, compounds that are more reduced than carbon dioxide and contain no carbon-carbon bonds; such compounds are termed C1 compounds. This statement defines the scope of this review. Specifically excluded from consideration here are the anaerobic methane producers and acetate producers, leaving the following two groups of C1 utilizers. (0) Methylotrophs, as defined by Colby & Zatman (39) and Quayle & Ferenci ( 158), can obtain their energy from the oxidation of C1 growth substrates and assimilate carbon as formaldehyde or as a mixture of formaldehyde and CO2, but always by pathways that are distinct from the Calvin cycle. These constitute the largest group. (b) The second group is CI-utilizing microor ganisms that oxidize C1 growth substrates to CO2 (which in most cases provides energy for growth) and then assimilate the CO2 so formed. These organisms may be conveniently labeled CI-utilizing autotrophs, bearing in mind that "autotroph" is used here sensu Schlegel (179) and Quayle & Ferenci (158) and not in the broader sense used by Whittenbury & Kelly (216). Recently, the observation of ribulose diphosphate carboxylase and phos phoribulokinase in a methane-oxidizing bacterium, Methylococcus capsula tus (Bath) (202), an organism previously considered a methylotroph, has opened up the possibility that some C1 utilizers might use both methylo trophic and autotrophic modes of metabolism simultaneously. However, in the absence of further evidence to the contrary, other C1 utilizers are assumed to fall into one or the other of the above two categories. THE TAXONOMY AND BIOLOGY OF METHANE-OXIDIZING MICROBES

Taxonomy and Nomenclature The present position on nomenclature and taxonomy of the methane oxidiz ers is confused, partially from the lack of information about taxonomically useful characteristics, and partially because of the absence of generally accepted taxonomic guidelines. Attempts are now being made to rationalize the situation, a recent and useful contribution being that of Romanovskaya, Malashenko & Bogachenko (1 67). They have reviewed the information about genera and species and have corrected nomenclature where it did not follow the Project for an International Code of Bacterial Nomenclature; the result is a collation of species descriptions, species considered to be incertae sedis for one reason or another, and a diagnostic key. Some elements in their conclusions are debatable, but their contribution will be invaluable in future


C1 METABOLISM

483

work leading to the publication of a widely acceptable taxonomy and no menclature of methane-oxidizing bacteria. There is a general consensus that the scheme of Whittenbury, Phillips & Wilkinson (2 17) should be the basis of a formally published taxonomy; in this scheme obligate methane-oxidiz ing bacteria are divided into two groups according to a variety of character istics. These include ultrastructural and other morphological features, type of resting cell formed, and physiological and biochemical properties. More recent work appears to support this basic grouping, but it has indicated a necessity for subgroupings-still above the level of genera-which as yet are not easily definable. A scheme shown in Table 1 identifies the subgroups referred to; it also incorporates both obligate and facultative methane-oxidizing bacteria. However, the validity of such a scheme can only be judged when a detailed comparative study of all strains has been undertaken. Reference to new features of the scheme are made in the text. Of the more recently described new types of methane oxidizer, two are worth highlighting: the facultative methane-oxidizing yeasts and the facul tative methane-oxidizing bacteria. Four different strains of yeast, the first known eukaryotes to oxidize methane, were isolated and described by H. Wolf and R. S. Hanson (personal communication). The organisms varied in their optimum growth temperature, maximum growth temperature, whether or not vitamins were required for growth, whether sugars were Table 1

Tentative classification scheme for methane-oxidizing bacteria3 Type I

Determinants

Type II

Membrane arrangement

Bundles of vesicular disks

Resting stages

Cysts

Major l:arbon assimiltation

RMP (3-hexulosephosphate synthase)

Serine pathway (hydroxypyruvate reduc

TCA cycle

Incomplete (2-Qxoglutarate dehydro

Complete

Nitrogenase

Some +

Paired membranes around cell periphery

(Azotobacter·like)

pathway

Exospores or lip id cysts

tase + 3-hexulosephosphate synthase) genase negative) + (those tested)

Predominant fatty acid

C chain length

Autotrophk CO2 fixation

DNA basc ratio (%G+C)

Isoeitrate dehydrogenase

50-54

NAD or NADP dependent

Cell shape

18

16 Subgroup A

Rod & ? coccus

Subgroup B

62.5

62.5+ (where tested)

NAD depen·

NADP dependent

dent

Coccus

some +

+

Examples

Methylomonas

Methylococcus

methanica and

Subgroup facultativec

+

Growth at 45'C

Methylomnnas a/bus

b Subgroup obligate

capsuJatus

Rod and vibrio Methanomonas methano-oxidans. Methylosinus trichosporium (both obligate) and

Methylobacterium organophilum (facul

tative) a Not all strains classifiable into type I and type II have been shown to possess all the biochemical characteristics outlined in this scheme.

bUse methanol and formaldehyde as carbon and energy source, but not C2+ compounds.

cUse variety of organic compounds, e.g. glucose as carbon and energy source.

484


fermented or used aerobically only, the types of sugars used, and the types of other organic compounds serving as carbon and energy sources. All were budding yeasts, forming bright pink colonies on yeast extract, peptone agar. Apart from the unequivocal demonstration that methane served as an energy source, little has been elucidated about the enzymes concerned. Recently, the ability of these yeasts to grow on methane has been confirmed in this laboratory.


Two reports of facultative methane-oxidizing bacteria, the first by Patt et al

(150)

and the second by Patel, Hou & Felix

organisms named as strains of a new genus,

(149), describe similar Methylobacterium. From the

information available (the organisms have an internal membraneous system of paired membranes when grown on methane, a serine type pathway of carbon assimilation, and a complete tricarboxylic acid cycle) they appear to be facultative varieties of the type II obligate methane oxidizers cate gorized as

''Methylosinus trichosporium" (217).

That they probably also

are exospore formers is indicated very clearly by an electron micrograph of a thin section shown in the paper by Patel et al

(149).

Substrates other

than methane utilized by these organisms include glucose, ethanol, ace tate, and succinate. Growth also occurs on nutrient agar and in nutrient

broth.

One advantage these organisms seem to offer over the obligate methane oxidizers in the study of methane oxidation relates to their mutability. R. S. Hanson (personal communication) has noted that it is possible to obtain mutagenization of these organisms growing on glucose and other organic substrates by techniques that do not lead to the successful isolation of mutants of these strains and obligate methane utilizers when growing on methane. Apparent resistance to mutagenization by methane oxidizers growing on methane has been studied by Williams & Shimmin

(218),

who

noted, for instance, that such organisms are probably nonmutable by

UV,

as the strain they studied lacked error-prone "SOS" DNA repair mecha msms.

Intracytoplasmic Membranes To date, only two categories of membrane arrangement have been identified

(52).

Indications that variants of these patterns occur seem not to have

excluded the possibility of faulty embedding techniques or that degenerate cells have been studied. On the other hand, the detailed freeze etching studies of Weaver & Dugan

thylosinus trichosporium Whittenbury (52).

(214)

on the membrane patterns of

Me

confirm and extend the description of Davies &

The most recent detailed study on the affect of growth conditions on

intracytoplasmic membrane development is that of Patt & Hanson

(151)


C1

METABOLISM

485

concerning the facultative species Methy!obacterium organophilum,' two points they noted were that only a cytoplasmic membrane was formed when the organism was grown on glucose or methanol, and under high oxygen tension, methane-grown organisms had approximately half the intracyto plasmic membrane content (as judged by membranes seen in thin sections and change in total lipid content) of methane-grown organisms cultivated under low oxygen tension. With a type I strain (Me thyloc occ us capsulatus), however, Linton & Vokes (1 1 8) noted that the membraneous develop ment observed in methane-grown cells persisted when the cells were grown on methanol [as noted previously by Davies & Whittenbury (52)].

Phospholipid and Fatty Acid Content Several studies on these lipids have been reported (e.g. 1, 124, 1 5 1). Makula ( 124), for instance, noted differences in the phospholipid content of type I and type II obligate methane oxidizers, as well as similarities. Most signifi cantly from a taxonomic point of view, he found that the type I species he examined, Methylococcus capsulatus, possessed an esterified fatty acid con tent of the CI6:0, C16:1 type, whereas the type II strain, Methylosinus trichos porium, possessed esterified fatty acids (up to 90% of the total content) of C1S:1 type. Andreev, Trotsenko & Galchenko (1) studied many strains and reported similar results with regard to the different types of fatty acid present in type I and type II strains. Patt & Hanson ( 1 5 1) found no usual or unusual lipid components to be associated with intracytoplasmic membranes that were not also associated with the cytoplasmic membrane. They also found squalene, previously associated only with M capsu!atus (22) of the methane oxidizers, to be present in Methylobacterium organophilum.

Tricarboxylic Acid Cycle A key difference between type I and type II methane oxidizers is the presence of a complete cycle in the latter whereas the former possess no detectable 2-ketoglutarate dehydrogenase (5 1). Recent studies on the in corporation of 14C-Iabeled acetate and the subsequent distribution of the label in amino acids of type I and type II organisms (147, 21 1) confirm the absence of 2-ketoglutarate dehydrogenase in type I strains and its presence in type II strains. What still remains unclear, however, is the reason for obligateness of the type II organisms.

Carbon Assimilation Pathways When growing on CI carbon compounds, the methane-oxidizing bacteria are presumed to assimilate most of their carbon by either the ribulose monophosphate (RMP) pathway or by a variant of the serine pathway.


486


However some type I organisms (RMP pathway) have been shown to contain low levels of hydroxypyruvate reductase activity (167, 2 15) and serine-g1yoxylate transaminase activity (1 67), leading to suggestions (167, 2 1 5) that carbon might be assimilated by both RMP and serine pathways in such organisms. Bamforth & Quayle (16) are rightly critical of this interpretation and claim that the mere presence of hydroxypyruvate reduc tase is not an indication that the complete serine cycle is in operation in such organisms [though it should be noted that the initial screening of type I and type II organisms for the presence of these pathways was based solely on the detection of the presence or the absence of key enzymes of these path ways (1 14)]. Bamforth & Quayle (16) highlight the danger of making such presumptions on the basis of detecting the presence or absence of one or two enzymes of a pathway by failing to find a role for hydroxypyruvate reduc tase formed in Paracoccus denitrificans growing autotrophically (carbon dioxide being fixed via the Benson-Calvin cycle) on methanol. However, although there can be no justification for assuming the presence of a path way on the basis of a single enzyme, it needs emphasizing, nevertheless, that there seems to have been no attempt to determine whether or not the RMP and serine pathways are present in the one organism. Merely assuming that because one major pathway is present (e.g. 1 6) there logically cannot be another as well is unreasonable. In our laboratory, for instance, Taylor (202) has shown that Methylococcus capsulatus (but no other methane oxidizer), which has the RMP pathway of carbon assimilation, also pos sesses phosphoribulokinase and ribulose diphosphate carboxylase, key en zymes of the Benson-Calvin cycle. The only assumption that can be made in this instance is that carbon dioxide can be fixed by the autotrophic mechanism, although it is not possible yet to estimate the significance of this activity in the overall carbon economy of this organism, or the environmen tal circumstances that dictate the expression and function of these auto trophic enzymes. Studies by H. L. Reed in this laboratory, briefly reported by Whittenbury et al (2 15) and results of S. Taylor (personal communica tion) point to the possible existence and role for a serine pathway in M capsulatus in relation to the oxygenase activity of ribulose diphosphate carboxylase-that is, in the assimilation of resultant phosphoglycollate. Although a serine pathway may appear to play only a minor role in carbon assimilation at best, such a role could be of significance in certain environ mental conditions [e.g. high oxygen tension, the availability of an energy source that is not a carbon source (215), and low carbon dioxide tension] and could be of crucial importance to the survival of the organisms in these circumstances. A complete study has yet to be made of this possibility. In this particular context, it may be that the apparently mystifying presence of hydroxypyruvate reductase (16) in P. denitrificans can also be explained.

C1 METABOLISM

487

Evidence of the ability of this organism to metabolize glycollate exists (e.g.

44, 136), though not necessarily via a serine pathway. However, as formal

dehyde is available to the organism (a consequence of the oxidation of methanol to carbon dioxide) under the conditions of growth where constitu tive hydroxypyruvate reductase activity is detected (16), it is not beyond question that Bamforth & Quayle (16) have overlooked a possible role for


their "transvestite" enzyme, albeit expressed to a significant purpose only

in special, as yet undefined, environmental circumstances (e.g. high oxygen low carbon dioxide tension) not tested by them. Perhaps it is the consider ation of different environmental conditions under which these organisms may have to survive from time to time in their natural environment that will lead to a greater understanding of the carbon assimilation routes of both methylotrophs and autotrophs.

The Relationship of Methylotrophy to Autotrophy Bacteria assimilating carbon via

C1 pathways have been compared over the

past few years with the aim of elucidating possible evolutionary relation ships and identifying the more important elements of their modes of life,

e.g. which of the various characteristics of autotrophs are the significant ones and do these lead to a broadening or narrowing of the concept of autotrophy? Two standpoints have emerged in this debate. One is that exemplified by Whittenbury & Kelly (216), who compared and contrasted methylotrophy and autotrophy and concluded that the concept of autotro

phy might be usefully extended to include all organisms able to assimilate C1 compounds as their sole carbon source. Such an assumption, they in dicated, would lead to a more revealing analysis and understanding of the concepts of autotrophy and methylotrophy and would focus upon specific

areas of interrelationships between the two categories of organisms. The second standpoint is a more cautionary and conservative one and is exem

plified by Quayle & Ferenci (158), who fear that the approach of Whitten bury & Kelly (216) could lead to an over-simplification of the issues, with

analogies being too easily drawn from similar-looking pathways. They (158)

appear to attach great significance to what they term "important biochemi cal pathways." Perhaps these two viewpoints represent, on the one hand, that of microbiologists concerned primarily with modes of life under vari ous environmental circumstances, rather than being greatly influenced by the apparent (at the time) importance of a particular biochemical pathway,

and on the other hand, that of biochemists more concerned with the pre sumed importance of a biochemical route expressed under particular condi

tions, and how it might have evolved, rather than with less-well-defined, loosely connected properties that go to make up a microbiological concept. Either standpoint will only prove to be of merit if it leads to a better

488


comprehension of methylotrophy and autotrophy. In favor of the Whitten bury & Kelly

(216) approach, thylococcus capsulatus has now

it is perhaps worth reiterating that

Me

been shown to possess key Benson-Calvin

carbon dioxide-fixing enzymes, in addition to the

RMP pathway,

and that

a serine pathway may play a role in the recycling of phosphoglycollate formed by the oxygenase activity of ribulose biphosphate carboxylase in the


organism, as it might also in Paracoccus denitrificans in which the presence of hydroxypyruvate reductase mystified Bamforth & Quayle

( 1 6).

In other

words, if methylotrophy and autotrophy are viewed as interrelated modes

of existence, then in addition to the many other similarities already in

dicated it becomes feasible to see such organisms as a collection of microbes able to assimilate carbon solely from C1 carbon compounds, varying in whether they use one, two, three, or (including the phosphoenolpyruvate carboxylase) four assimilation routes, with the last never being the only route of assimilation.

MICROBIAL ASSIMILATION OF CI COMPOUNDS Three mechanisms for the assimilation of C1 compounds are recognized: the

ribulose diphosphate pathway (Calvin cycle) of CO2 assimilation, the RMP pathway of formaldehyde assimilation, and the serine pathway. In the past

it has been taken for granted that a particular C1-utilizing microorganism possesses one or another of these pathways to the exclusion of the other two

(2, 157).

Sufficient evidence has now accumulated, however, to foster the

suspicion that some microorganisms may use more than one mechanism for

C1 assimilation either under different growth conditions or indeed simulta neously. This evidence can be summarized as follows.

1. Some organisms that apparently use the RMP pathway as their major

mechanism for C1 assimilation contain low to moderate specific activities of hydroxypyruvate reductase (BC

1.1.1.29), an enzyme normally asso (97, 1 14, 163, 1 82, 199). Other enzymes of the serine pathway may also be present, as in methanol-grown Streptomyces sp. (97) or methane-grown Methyloccocus capsulatus (Texas) and Me thylomonas methanica (174). Indeed radiotracer studies may indicate some

ciated with the serine pathway

initial incorporation of C1 compound into intermediates other than sugar phosphates, as in methane-grown M.

2.

Extracts of

capsulatus (Bath) ( 1 63). Paracoccus denitrificans and Thiobacillus novellus contain

hydroxypyruvate reductase activity that is constitutive and of unknown metabolic function

( 1 6, 3 1),

in addition to enzymes of the autotrophic

ribulose diphosphate pathway. From these observations, Bamforth &

Quayle ( 16) concluded that the presence of hydroxypyruvate reductase activity in a Crutilizing microorganism could no longer be accepted as indicating the presence of the serine pathway.


C, METABOLISM

489

3. Taylor (202) has demonstrated the presence of ribulose 1 ,S-diphos phate carboxylase (BC 4. 1 . 1 .39) and phosphoribulokinase (Be 2.7. 1 . 1 9), key enzymes of the ribulose diphosphate pathway, in extracts of methane grown M. capsulatus (Bath). In vivo fixation of CO2 during growth on methane was also demonstrated (203). 4. Enzymic analysis and radiotracer studies have shown that Pseudomo nas gazotropha uses the ribulose diphosphate pathway during growth on CO whereas the serine pathway is involved in methanol assimilation (168). There is no convincing evidence as yet for the simultaneous operation of two complete CI assimilation pathways in any microorganism. However, it is clear that certain CI utilizers, while using one pathway as the major source of fixed carbon, have the ability to assimilate small amounts of C1 compound by a different route (other than the usual heterotrophic carboxy lation reactions). M capsulatus (Bath) would seem to be the best example of such an organism.

Ribulose Diphosphate Pathway of CO2 Assimilation More than a dozen microorganisms assimilate reduced CI compounds by this familiar autotrophic pathway (Table 2) following their oxidation to CO2, despite the considerable savings in energy offered by the alternative RMP or serine pathways (3, 1 58). Many of these organisms are normally considered to be COrutilizing phototrophs or chemolithotrophs for which the acquisition of the few enzymes required to oxidize C1 compounds to Table 2

Microorganisms that assimilate reduced C 1 compounds via the ribulose diphos

phate cycle of CO2 fixation Organism

C I growth substrate

Pho totrophs Rhodopseudomonas palustris (155)

formate

Rhodopseudomonas acidophila (153, 169)

methanol; formate

Spirulina platensis (32)

CO

Chemolithotrophs

Hydrogenonomonas eutropha (138) Alcaligenes FORI (30) Pseudomonas gazotropha (168)

Pseudomonas carboxydovorans (134) Paracoccus denitrijicans (44, 183)

formate formate CO CO methanol; formate

Microcyclus aquaticus (138)

methanol; formate

Thiobacillus novel/us (31)

methanol; formate; formamide

Others

Bacterium formoxidans (188)

formate

Pseudomonas oxalaticus (159)

formate

Bacterium sp. 7d (123)

methylamine


490


CO2 confers a bonus in terms of nutritional versatility (169). For example, many strains of the photosynthetic purple non-sulfur bacteria photoassimi late methanol and/or formate anaerobically when these are provided in place of or in addition to bicarbonate (ISS, 162). Recently, following the discovery that the methanol dehydrogenase (EC 1.1.99.8) of Rhodop seudomonas acidophila is oxygen sensitive (169), the growth of this organ ism on methanol aerobically and in the dark has been achieved by using reduced oxygen tensions (153). Pseudomonas oxalaticus, which is neither a phototroph nor a chemoli thotroph, also assimilates formate via the ribulose diphosphate pathway (156). This contrasts with the metabolism of formate by Hyphomicrobium X (10) and Pseudomonas AM I (112); the latter organisms assimilate for mate via the serine pathway following its reduction to N-5·1O-methylenetet rahydrofolate. P. oxalaticus oxidizes formate by a soluble NAD-dependent formate dehydrogenase (EC 1.2.1.2), yielding NADH and hence ATP, whereas the CO2 so generated is assimilated. Oxalate is also a growth substrate and its energy metabolism is similar. Oxalate is first decarbox ylated to formate by energetically neutral reactions involving coenzyme A (CoA) thioesters as intermediates, and then oxidized by formate dehydroge nase (160). Oxalate carbon, however, is assimilated by the glycerate path way after reduction to glyoxylate, with the enzymes of the ribulose diphosphate pathway being "switched off" during growth on oxalate (161). P. oxalaticus is therefore an ideal organism in which to study the regulation of the ribulose diphosphate pathway. Such studies were started by Black more & Quayle (23) and recently were extended by Harder and his col leagues (58-61, 103). Catabolite repression of the autotrophic enzymes, rather than their induction by formate, appears to be the likely control mechanism (103), in keeping with results obtained earlier with Rhodospiril lum r ubrum (184). RMP Pathway

RMP, a pathway of formaldehyde assimilation (Figure 1), was proposed by Kemp & Quayle (99) when short-term incubations of the methane-oxidizing bacterium Methylomonas methanica with 14C-labeled methanol or formal dehyde resulted in early labeling of sugar phosphates, particularly hexose phosphates. Subsequent work (98, 113) showed the key reactions to be those catalyzed by 3-hexulose phosphate synthase and phospho-3-hexuloisome rase, respectively (Figure 2). These two key enzymes of the RMP pathway, in many cases together with other evidence for the presence of the pathway, have since been found in other methylotrophs that cannot use methane (Table 3). The enzyme 3-hexulose phosphate synthase has been purified from four methylotrophs (71, 94, 171, 187).

491

C1 METABOLISM

MP Xu5P

�:O AT!y�

Xu5P

"'�b "Y) DH� 33P "

f6Pf1!LFOP

CHAP G3P DHAP

G3P

�


G3P

E..u.twive PHIt. Vgrjgnt of

yrp

2

SOP

Entner_Poudo(Qff Variant of S'99W

F6P

�

GJP

Variant of Stagu

S'ogft...!.U

FOP

"---. ----? .

DHAP

3HCHO.2ATfl--+DHAP+ 2ADftt.P j

Stage 2 3HCHQ+ NAO-.PYR+NADH2

Figure 1

RMP and its variations. Ru5P, Ribulose-5-phosphate; Hu6P, D-erythro-L-glycero-

3-hexulose-6-phosphate; F6P, fructose-6-phosphate, FDP, fructose-l,6-diphosphate; G3P,

glyceraIdehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; E4P, erythrose-4-phos phate; Xu5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; SDP, sedoheptulose-I ,7diphosphate; R5P, ribulose-5-phosphate; G6P, giucose-6-phosphate; 6PG, 6-phosphogIu

conate; pyr, pyruvate; 1, 3-hexolosephosphate synthase; 2, phospho-3-hexuloisomerase; 3,

6-phosphofructokinase (Ee 2.7.1.11); 4, fructose diphosphate aldolase (Ee 4.1.2.13); 5, tran sketokase (Ee 2.2.1.1); 6, transaldolase (Ee 2.2.1.2); 7, ribulose phosphate epimerase (Ee 5.1.3.1); 8, ribulose phosphate isomerase (Ee 5.3.1 .6); 9, sodoheptulose diphosphatase; lQ, fructose diphosphatase (Ee 3.1.3.11); 11, triosephosphate isomerase (Ee 5.3 . 1 .1) ; 12, glucose phosphate isomerase (Ee 5.3.1 .9); 13, glucose-6-phosphate dehydrogenase (Ee 1 .1 .1 .49); 14,

6-phosphogluconate dehydratase (Ee 4.2.1.12) + phospho-2-keto-3-deoxygluconate aldolase

(Ee 4.1.2.14); 15, transketolase + triokinase (Ee 2.7.1.30).

�H20H

I

CO J

I

HCOH I

CH20P

I

CO I

Figure 2

�

I

HO CH ,

HCOH

HCOH

HCOH

HCOH

J

I

CH20P Ru5P

CO

HOCH )

HCHO+ HCOH

�H20H

CH20H

Hu6P

I I

CH20P F6P

Key reactions of the RMP. See Figure 1 for abbreviations.

492

COLBY, DALTON & WHITTENBURY Table 3 Methylotrophs using the RMP Methylotrophs

Ref.

Methane-oxidizing obligate

Methylococcus capsulatus ( Texas)a,b

b M. capsulatus (Bath) ,

M. ucrainicusc, d


M. thermophilus c ,

c

d

Methylomonas methanicaa, b, d

M. agileC

M. rosaceusc

M. rubrumc, d Methylobacter capsulatusC

M. mininusc M. bovisc,

d

M. vinelandiic, d

1 13, 199 163,215 182 182 99, 199 114 114 182 114 114 182 182

Other obligate

Bacterium 4B6a

43

Bacterium C2A 1 a

43

Pseudomonas WI c Methylomonas M15c

Methylophilus methylotrophusa Methylomonas aminofaciens 7 7ac

Pseudomonas W6a L3c

Facultative (not methane oxidizing) Bacillus spp. PM6 + S2A I a Bacterium W3A 1 a Arthrobacter 2B2a Arthrobacter globiformis B-175 Pseudomonas CC

Pseudomonas oleovoransb, Streptomyces sp. 239c, d

c

92 171 200

94 12 91 43 43 43 121 80 1 19,122 97

aHexulose phosphate synthase and at least some other enzymes of path way demonstrated. bIn vitro radiotracer studies indicate assimilation of C 1 compound by pathway. COnly hexulose phosphate synthase activity demonstrated in vitro. dOne or more enzymes of serine pathway also present.

The RMP pathway is conveniently divided into three stages (Figure 1). Stage 1, the condensation of three molecules of formaldehyde with three molecules of ribulose-5-phosphate to yield three molecules of fructose-6phosphate, is common to all methylotrophic bacteria using the pathway. Stage 2 involves the splitting of one molecule of fructose-6-phosphate to produce two C3 compounds. In some methylotrophs, such as Bacill us spp. PM6 and S2Al (43) and Arthrobacter globiformis B-175 (123), this is achieved by enzymes of the glycolytic sequence as originally envisaged by


C1 METABOLISM

493

Kemp & Quayle (99). In other methylotrophs, such as bacteria 4B6, C2AI, and W3Al (43), Pseudomonas W6 (12), and Pseudomonas oleovorans (123), this split is catalyzed by enzymes of the Entner-Doudoroffpathway and the analogous enzymes of the glycolytic sequence are absent. The methane oxidizers M methanica and Methylococcus capsulatus (199) contain en zymes of both sequences and their relative importance is not known. Stage 3 involves the regeneration of three molecules of ribulose-5-phosphate from the two molecules of fructose-6-phosphate and one molecule of glyceralde hyde-3-phosphate produced in stages 1 and 2. These sugar phosphate inter conversions are catalyzed by transaldolase and transketolase in bacteria 4B6, C2Al, and W3Al (43) and in M methanica and M. capsulatus (199). Bacillus spp. PM6 and S2Al, however, lack transaldolase and their sugar phosphate interconversions involve sedoheptulose-l,7-diphosphatase and fructose diphosphate aldolase instead (43). Other methylotrophs have not been examined for modifications in stage 3 of the pathway. The resemblance between the autotrophic ribulose diphosphate pathway and the RMP pathway is self-evident. In a recent review Quayle & Ferenci (158) have attempted "to present a logical sequence of chemical and bio chemical evolution which might have led from primeval soup chemistry to the RuDP cycle by way of the RuMP (sic.) cycle." The reader is referred to this article for a thorough discussion of this area.

Do Methanol-Utilizing Yeasts Use a Modified RMP Pathway? Incubation of methanol-utilizing yeasts with 14CH30H results in the eady labeling of sugar phosphates (75, 76); similar results were obtained with the methanol-utilizing mycelial fungus Trichoderma lignorum (208). However, attempts to demonstrate 3-hexulose phosphate synthase activity in cell-free extracts of yeasts resulted in low or zero activities that when present were often dependent on the presence of A TP (29, 54, 76, 172). In an attempt to resolve this unsatisfactory situation, van Dijken and his colleagues re cently investigated the assimilation of methanol in two yeasts, Hansenula polymorpha and Candida boidinii (55). Following an earlier suggestion (46) that transketolase might catalyze a pentose-phosphate-dependent fixation of formaldehyde, yielding dihydroxyacetone and glyceraldehyde-3-phosphate (see Figure 1), these workers formulated a modified pathway they termed a dihyroxyacetone pathway. The presence in extracts of C. boidinii of fructose diphosphase activity and of a triokinase capable of phosphorylating dihydroxyacetone and that is induced during growth on methanol (55) is consistent with the operation of such a pathway, although conclusive proof of its validity is not yet forthcoming.

494


The Serine Pathway Short-term incubations of Pseudomonas AMI with 14C-Iabeled methanol resulted in early labeling of serine followed by glycine, malate, and aspartate (109). The distribution of label from 14C-Iabeled methanol or 14C-labeled bicarbonate in these early labeled intermediates was consistent with the important C1 fixation reactions being


serine transhydroxymethylase,

(0)

the fixation

of formaldehyde by

HCHO + THF � N-5 IO meth ylene-THF (chemical) glycine + N 5 lo-meth ylene THF � s erin e + THF, ,

-

-

.

-

1.

where THF is tetrahydrofolate, and (b) the formation of malate by carboxy lation of a C3 compound derived from serine (110). Subsequently, the latter reaction was found to be catalyzed

by phosphoenolpyruvate carboxylase

(111), phosphoenolpyruvate

+ CO2 + H20

�

oxaloacetate + Pi.

2.

and many of the intervening reactions between serine and oxaloacetate were Pseudomonas AMI (24, 112). Further labeling experiments using [2,3-14C2] succinate (176) confirmed that the glycine C1 acceptor was regenerated by cleavage of a C4 compound (presumably derived from oxaloacetate), thus completing a cyclic serine pathway for the formation of a C2 compound from formalde hyde and CO2 (Figure 3). The crucial C cleavage reaction was first described in two methylamine 4 utilizing methylotrophs, Pseudomonas MA (85) and bacterium 5H2 (45). The enzyme responsible was called malate:ATP lyase (cf 20 7) and it cat alyzed the following reaction: malate + ATP + CoA ---+ glyoxylate + acetyl-CoA + ADP + Pi' Subsequently, this reaction was discovered to be separable into two reactions catalyzed by malate thiokinase,

identified by enzymological studies with cell-free extracts of

malate + ATP + and malyI-Co

CoA � malyl-CoA + ADP + Pi,

3.

A lyase,

malyl-CoA ---+ glyoxylate + acetyl-CoA,

4.

respectively (84). Only malyl-CoA lyase has been found in Pseudomonas AMI (174) and the mechanism for making malyl-CoA in this organism is still unknown. Nevertheless, recent studies have confirmed the importance of malyl-CoA lyase during the growth of Pseudomonas AMI on C1 com pounds (175). As presented in Figure 3, the serine pathway involves some mechanism for the oxidation of acetyl-CoA to glyoxylate, which on transamination

C, METABOLISM

495

HCHO (x2)

glycine� (x2) serine (x2)

b� b

FAD succinate �9IYOXYlate (x2) � FADH2 'jV j I i so e rate Il + I L Iate � Ilj NAD+� /�aeetyl-CoA NADH+H+ OAA /'


I

{

\

OHPYR (x2) rNADH+H+ .['--NAO+ G)�ATP 'b-AOP 2-PG:� 1 �3-PGA PEP e

f

r C02

malyl-CoA OAA + g LNADH+H AOP� h ATPA malateANAo+ .

Figure 3

Serine pathway.

a,

Serine transhydroxymethylase (BC 2.1.2.1); b, serine glyoxylate

amino-transferase; c, hydroxypyruvate reductase (BC 1.1.1.29); d, glycerate kinase (BC 2.-

7.1.31); e, phosphopyruvate hydratase (BC 4.2.1.11); t phosphoenol-pyruvate carboxylase (BC 41.1.31); g. malate dehydrogenase (BC 1.1.1.37); h. malate thiokinase (BC 6.2.1.-); i,

malyl-CoA lyase (EC 4.1.3.24); j. isocitrate lyase (EC 4.1.3.1); - - - - -, unknown reactions;

OHPYR, hydroxypyruvate; GA, glycerate; PGA, phosphoglycerate; PEP, phosphoenolpyru vate; OAA, oxaloacetate. Net reaction �

3-PGA

+

(IL+): 2HCHO + CO2 + FAD + 2NADH2 + 3ATP

2ADP + 2 NAD + FADH2•

regenerates the second molecule of glycine acceptor; this allows a C3 or C4 compound to be withdrawn from the cyclic pathway for assimilation into cell substance. In some methylotrophs that use the serine pathway this mechanism involves isocitrate lyase together with some enzymes of the tricarboxylic acid (TCA) cycle as shown in Figure 3. This isocitrate lyase positive (IL +) serine pathway has been reported in Pseudomonas MA (85), Pseudomonas aminovorans (15, 108), bacterium 5H2 (45), and Pseudomonas MS (212). Bacterium 5BI is unusual in containing high spe cific activities of isocitrate lyase during growth on acetate, but not during growth on a CI compound, trimethylamine (39, 47). Related to this observa tion is that of Bellion & Woodson (20), who found that the isocitrate lyase present during growth of Pseudomonas MA on methylamine was distinct from the isofunctional enzyme found during growth on acetate. These authors suggested that this allowed for independent regulation of isocitrate


496

COLBY, DALTON & WHITIENBURY

lyase activity during growth on acetate, when isocitrate lyase is involved with malate synthase (BC 4.1.3.2) in a complete glyoxylate cycle, and on C1 compounds when malate synthase is repressed. Many methylotrophs that use the serine pathway (see Table 4), including Pseudomonas AMI (19, 65), do not contain significant specific activities of isocitrate lyase during growth on their C1 substrates. This IL- serine pathway (Figure 3) has been extensively studied in Pseudomonas AMI by Anthony (2). The results of growth and radiotracer studies, using mutants Table 4

Methylotrophs that use the serine pathway

Ref.

Methylotrophs Methane-oxidizing ob ligate

Methylosinus trichosporiuma

114

M. sporiuma

114

Methylocystis parvusa Methanomonas methanooxidansa, Methane-oxidizing facultative

Methylobacterium organophiluma, Isolate R6a,

c

c

c

114 113

143 149

Other facultative

Pseudomonas AM1a-c

Pseudomonas 3A2a Pseudomonas MAa-c

109-112 39 85

Pseudomonas MSa, c

103 ,212

Pseudomonas sp. 20

123

Pseudomonas spp. 1 + 1 3 5 a Pseudomonas YRa

Pseudomonas TPI a

Pseudomonas PCTNa

166

19 19 19

Pseudomonas aminovoransa, c P. gazotrophaa, b

108

P. methylica

123

Organism J B1a Organism FWCa Organism PARa,

168 19

c

Bacterium 5 Bl a

19 19 39

Bacterium 5H2a, c

45

Streptomyces spp. 239a-d

97

Hyphomicrobium spp.a-c

9

Gliocladium deliquescensa-c

173

Paecilomyces cariotia-c

173

aHydroxypyruvate reductase (HPR) activity demonstrated in vitro. bIn vivo radiotracer stu dies indicate assimilation of Cl compound b y serine pathway. cHPR and at least some other enzymes of the pathway demonstrated. dSome enzymes of RMP also present.


C1 METABOLISM

497

that cannot grow on C1 and/or C2 compounds, suggest that Pseudomonas AMI contains enzymes that oxidize acetyl-CoA directly to glyoxylate, probably with glycollate as an intermediate. These enzymes are also impor tant during the growth of Pseudomonas AMI on C2 compounds such as ethanol and acetate (Pseudomonas AMI does not synthesize isocitrate lyase during growth on C2 compounds either) and even during growth on some C3 compounds. The biochemistry of this direct acetate oxidation pathway is presently obscure. Genetic Regulation of CrMetabolism Coordinate induction or repression of enzymes involved in C1 metabolism has been observed in three methylotrophs that use the serine pathway, viz Pseudomonas AMI (66), Methylobacterium organophilum (143), and Pseudomonas aminovorans (26). In Pseudomonas AMI the enzymes in volved are all from the serine pathway, but in M organophilum and P. aminovorans enzymes from the serine pathway and from C1 oxidation pathways are synthesized coordinately. Further evidence for coordinate expression has come from the isolation of pleiotropic mutants of M organophilum (143) and P. aminovorans (15) deficient in several enzymes involved in C1 metabolism. Transformation studies with mutants of M organophilum have shown physical linkage between many of the genes involved. (144). MICROBIAL OXIDATION OF C1 COMPOUNDS

In most instances microbial growth on C1 compounds does not involve the central catabolic pathways generally associated with the energy metabolism of aerobes. Evidence in support of this statement comes from the repressed specific activities of TCA cycle enzymes found in many facultative methyl amine- or methanol-utilizing bacteria during growth on C1 compounds compared with those found during growth on CrC6 substrates (39, 48,201), and the low or zero specific activities of at least some TCA cycle and related enzymes found during the growth of other microorganisms on C1 com pounds (7, 8, 11, 20, 39,42, 48,51). The methylamine utilizer Pseudomonas MA is exceptional in containing high activities of TCA cycle enzymes during growth on a C1 substrate and it has been suggested (140) that the TCA cycle is involved in energy conservation from C1 compounds in this particular organism. C1-utilizing microorganisms oxidize their growth substrates by the series of special C1 oxidation pathways shown in Figure 4. In this figure formalde hyde occupies a central position as a common intermediate in the oxidation of most C1 compounds.

498


Methane IMethanol


co

1

Oxidation Pathway

Methyl Amine Oxidation Formaldehyde

Pathway

Oxidation Pathway HCOOH

Figure 4

C. oxidation pathways. THF, Tetrahydrofolate.

Oxidation of Carbon Monoxide Measurements of carbon monoxide uptake in soils has indicated that the global rate for CO consumption by microorganisms is about 5 X 1014 glannum (116). This accounts for the transformation of about 50% of the total carbon monoxide produced in the environment. Despite the ubiqui tous distribution and biological consumption of carbon monoxide, early attempts to isolate microorganisms capable of growth at its expense were not very rewarding. The only successful isolate reported was by Kistner (101), who observed that the organism Hydrogenomonas carboxydovorans would grow aerobically at the expense of hydrogen and/or lactate and oxidize carbon monoxide to carbon dioxide. The ability of the organism to oxidize H2 and CO was rather unstable, and the possibility that the culture was a mixture of heterotrophic and autotrophic species or that the auto trophic characters were plasmid-borne as considered by Zavarzin & Noz hevnikova (225) will never be proved since the culture has subsequently been lost. Recently, Meyer & Schlegel (134) isolated an organism that closely corresponded to Kistner's (101) original description. This organism did not have an obligatory requirement for organic growth factors and hydrogenase was present whether the cells were grown on CO, CO2, or pyruvate. They named the organism Pseudomonas carboxydovorans (Kist ner). A number of authentic CO utilizers have now been isolated and classified (142, 177). They appear to be a very diverse group of organisms containing five species identified as Seliberia carboxydohydrogena. Pseudo-

C1 METABOLISM

499

monas carboxydoflava, Pseudomonas gazotropha, Comamonas compran soris, and Achromobacter carboxydus. All but the latter species could be classified as hydrogen bacteria since

they would grow under H2/C02/02 gas mixtures as well as CO/Oz mix tures. P.

gazotropha

can grow aerobically with methanol as the sole carbon


and energy source. Energy for growth under H�C02/02 mixtures is ob tained from the oxidation of hydrogen. Analysis of the early labeled prod ucts of 14COZ incorporation in Seliberia indicated that carbon dioxide was being fixed by the Calvin cycle (141). Only in P.

gazotropha,

however, have

both RuDP carboxylase and phosphoribulokinase activities been detected

(168), the kinase drogena (141).

apparently being absent from extracts of S.

carboxydohy

Growth under CO/Oz gas mixtures was reported as being much slower than growth with H2/Coz/Oz (doubling times of around

50 hr in the former

instance and 28 hr in the latter) (178). In a number of strains tested approxi mately 4% ofthe carbon monoxide was converted into cell biomass and the remainder was oxidized to CO2 (226). The mechanism whereby these organ isms convert the CO into assimilable carbon has been the subject of a great deal of speculation and only a limited amount of experimental verification.

The consensus of opinion is that the CO is first oxidized to COz, which is

then fixed autotrophically via the Calvin cycle. This ability to oxidize CO to CO2 is not restricted to the carboxydobacteria listed above, but it is also

observed, gratuitously, in anaerobes such as Methanobacterium ba,keri (102), Methanobacterium formicicum (102), Desulfovibrio desu/furicans (220-222), Clostridium pasteurianum (74, 204), Rhodopseudomonas (90, 209), Clostridium welchii (195), and Clostridium!ormicoaceticum and Clos tridium thermoaceticum (53), as well as in some methane oxidizers (36, 70, 72, 93). Only in the case of Rhodopseudomonas sp. strain 1 (209) was

carbon monoxide shown to be serving as the sole carbon and energy source

for growth after repeated subculture in a medium containing carbon mon

oxide and some vitamins. Methanobacterium thermoautotrophicum would grow in a bicarbonate-buffered minimal medium with carbon monoxide as the sole energy source, but the growth rate was only hydrogen was used

1%

of that when

(50).

Oxidation of CO to CO2 in the anaerobes is a strictly anaerobic process

both in vivo and in vitro and in the cases studied the source of oxygen for the oxidation is water. The in vitro electron acceptor can be a dye such as

methyl viologen, benzyl viologen, or methylene blue. Neither NAD(P)H

nor ferredoxins function in this respect, although in the case of M. ther moautotrophicum a fluorescent FMN-derivative (F420) acts as the acceptor

(50). A vitamin B12 compound has been implicated in anaerobic CO oxida (53, 73). The aerobic methane oxidizers can also use CO

tion by clostridia


500


gratuitously (36, 70, 72, 93). Studies with cell-free extracts revealed that the methane monoxygenase could be responsible for the incorporation of the second atom of oxygen into carbon monoxide from dioxygen (72). As with anaerobes, the methane oxidizers cannot grow at the sole expense of carbon monoxide. However, one report that Methylococcus capsulatus (Bath) con tains both RuDP carboxylase and phosphoribulokinase (202) opens up the possibility that such an organism could grow with CO as its sole carbon and energy source, particularly since the amount of energy available from the monooxygenase type reaction, i.e. NADH + H+ + CO + O2 -476 KJ/mol,

.6. Go1

-+

CO2 + H20 + NAD+ . . . . . . 5.

=

is far greater than that from the non-monooxygenase reaction CO + H20 .6. Go1

=

CO2 + 2H . . . . . . -20 KJ' mole-I of CO. -+

6.

There has only been one report of a cell-free system from an organism that can grow with CO as its sole carbon and energy source. In this organ ism (a hydrogen-utilizer strain 1 515 pI) ( 1 33) the oxidation of CO could be coupled to methylene blue reduction anaerobically, thereby discounting the possibility that CO was oxidized by a monooxygenase type reaction. Fur thermore, it was observed that formate oxidation coupled to methylene blue reduction was located in the same subcellular fraction as the CO-oxidizing enzyme. The possibility that formate was an intermediate in the reaction CO + H20 -+ HCOOH -+ CO2 + 2e + 2H+ was discounted based on inhibitor and kinetic studies, and it was concluded that the reaction was as reaction 6. More recently, Kirkonnell & Hegeman (100) used H2 1 80 and 1802 to determine the source of the second oxygen atom in CO2 and found that neither water nor dioxygen was the supplier and that other sources must be sought. -

Oxidation of Methane and Methanol Methane is used as sole carbon and energy source by a number of bacteria and a few strains of yeast (R. S. Hanson, personal communication). Little is known about the growth of yeasts on methane and therefore this is not considered further here. Our understanding of prokyarotic methane oxida tion, on the other hand, has advanced considerably since this was last considered (164). The oxidation of methane to carbon dioxide appears to proceed via a series of two-electron oxidation steps (Figure 5). Formalde hyde occupies a central position in the metabolism of methane since it is at this level that the carbon is both assimilated into biomass and dis similated to carbon dioxide to provide energy for growth. The majority of

C1 METABOLISM

r

Hp

HC O I

�

I I J. Annu. Rev. Microbiol. 1979.33:481-517. Downloaded from www.annualreviews.org Access provided by Technische Universiteit Eindhoven on 02/01/15. For personal use only.

501

2H

�

HCOO

02

NAO+ NAOH + H+

Ass i m i lat i o n Figure 5

Pathway for the bacterial oxidation of methane and methanol. I. Methane monoox

ygenase; 2, methanol dehydrogenase; 3, formaldehyde dehydrogenase; 4, formate dehydroge nase.

organisms that grow on methanol oxidize the substrate to carbon dioxide in a series of reactions similar to those given above. The oxidation of methanol in bacteria appears to be mediated by a methanol dehydrogenase, although in Methylococcus capsulatus the particulate enzyme does appear to have oxidase activity as well (2 10). In the methanol-utilizing yeasts growth on methanol is accompanied by a profound increase in the number of microbodies known as peroxisomes (57, 77, 78, 83, 170) compared with the number observed when the organisms are grown on glucose. These organelles contain high levels of fiavin-dependent alcohol oxidase and cata lase. The need for high levels of catalase in association with the alcohol oxidase becomes apparent when it is seen that the oxidation of methanol in yeasts produces formaldehyde and hydrogen peroxide. The latter is effectively removed from the cell by the action of the catalase. The formal dehyde thus formed is probably exported from the peroxisome and as similated via a RMP pathway (55), with dissimilation occurring via formaldehyde and formate dehydrogenases (77, 96).

Oxidation of Methyl Amines The methyl amines are a series of ammonia derivatives in which successive hydrogen atoms are replaced with methyl groups. The ability to utilize methyl amines as growth substrates is widespread among C I -utilizing mi croorganisms (Table 5). The methyl amine oxidation pathway comprises a series of oxidative N-demethylations by which successive C-N bonds in the substrate are cleaved and the methyl groups are released as formaldehyde. Hence trime thylamine, for example, is oxidized to 3 mol of formaldehyde and I mol of ammonia with dimethylamine and methylamine as intermediates. It is char acteristic of the pathway that for each oxidative N-demethylation step, alternative enzyme systems are found in different microorganisms. Another

502

COLBY, DALTON & WHITTENBURY Table 5 C rutilizing microorganisms that grow on methyl amines Organism C 1 -u tilizing au totrophs Paracoccus denitrificans Bacterium sp. 7d

Ref.

44

123


Obligate methylotrophs Bacterium 4 B6

40

Bacterium C2A l

40

Pseudomonas W I Methylomonas methylovoro

Pseudo monas J

48 1 04 1 25

Facultative methylotrophs Pseudomonas aminovorans

1 25

P. oleovorans

123

P. methylica

123

Pseudomonas AM I

152

Pseudomonas M 2 7

4

Pseudomonas 3A2

39

Pseudomonas MS

213

Pseudomonas M A

1 80

Pseudomonas RJ 1

1 29

Pseudomonas spp. 1 & 1 3 5

1 66

Bacterium 5 H 2

82

Bacterium 5 B l

40

Bacterium W3Al A rthrobacter B- 1 75

43 121

A rthrobacter 2 B 2

40

Bacillus PM6

43

Hyphomicrobium spp.

7

Trich oderma lignorum

208

notable feature of the pathway is that in most organisms it contains no NAD-linked dehydrogenases and in this respect it resembles the methane/ methanol oxidation pathway. Thus in the majority of methylotrophic bac teria and yeasts, reduced nicotinamide nucIeotides are generated only dur ing the oxidation of formaldehyde to CO2, In fact there are exceptions to this generalization. NADH is generated during the oxidation of trimethyl sulphonium by Pseudomonas MS (92), during the oxidation of methylamine to formaldehyde in Hyphomicrobium vulgare (120) and Pseudomonas me thylica ( 139), and possibly during the oxidation of methanol to formalde hyde in some yeasts (62, 1 30). OXIDAnON OF TETRAMETHYLAMMONIUM ION

Bacterium 5H2 oxi dizes tetramethylammonium chloride via trimethylamine, trimethylala mine N-oxide, dimethylamine, and methylamine, and the presence in crude

C1 METABOLISM

503

extracts of an inducible tetramethylammonium monooxygenase catalyzing reaction has been established (82).


(CH3)4N+ + NAD(P)H + H+ + O2 � (CH3)3NH+ + HCHO + NAD(P)+ + H20.

7.

The enzyme was unstable and although the activity could be resolved into two fractions, the components could not be completely purified (81). Activ ity was not inhibited by CO, suggesting that cytochrome P450 was not involved. OXIDATION OF TRIMETHYLAMINE Two mechanisms for the oxidative N-demethylation of trimethylamine occur among methylotrophic bacteria (40, 123). The direct route (reaction 8) catalyzed by trimethylamine dehy drogenase (BC 1 .5.99.7) was first described in bacteria 4B6 and C2Al (40, 41) and subsequently was found in bacterium W3A l (43), in Hyphomi crobium vulgare NQ + ZV (25, 123), and in Mycobacterium sp. 10 (123).

(CH3)3NH+ + flavoprotein + H20

(CH3)2NH2+ + HCHO + reduced flavoprotein.

�

8.

The natural electron acceptor has been purified from bacterium W3A l ( 1 89); it is a flavoprotein of molecular weight 77,000 that contains 1 FAD per mol. Other trimethylamine-utilizing bacteria oxidize trimethylamine by the sequential action of two enzymes viz trimethylamine monooxygenase (EC 1 . 14. 13.8) and trimethylamine N-oxide aldolase (demethylase) (EC 4. 1 .2.-) as follows (40, 43, 1 23, 25): (CH3)3NH+ + O2 + NADPH + H+

(CH3)3NO + H+ + H20 + NAD(P)+;

�

and (CH3)3NO + H+

�

(CH3)2NH2+ + HCHO.

Trimethylamine monooxygenase has been purified about six-fold from ex tracts of Pseudomonas aminovorans, but its instability so far has prevented its further purification (25). Trimethylamine N-oxide aldolase has been purified from Bacil/us PM6 ( 1 37). The enzyme is a single polypeptide of molecular weight about 45,000 that contains no known prosthetic group. Enzyme activity is stimulated by ferrous iron, glutathione, and L-ascorbate. A similar enzyme was partially purified from P. aminovorans (107). OXIDATION OF DIMETHYLAMINE With one exception, the oxida tion of dimethylamine in all methylotrophic bacteria that have been died is catalyzed by a dimethylamine monooxygenase (Be 1 . 14.99.-): (CH3)2NH2+ + O2 + NADH + H+ � CH3NH3+ + H20 + NAD+.


504


Dimethylamine monooxygenase has proved difficult to isolate (67), but recently a 126-fold purified preparation has been obtained from Pseudomo nas aminovorans (28). The enzyme is potently inhibited by carbon monox ide and its activity is thought to involve a P420-type cytochrome (27). A dimethylamine dehydrogenase activity that requires phenazine methosul fate as in vitro electron acceptor has been detected recently in crude extracts of Hyphomicrobium X (132, 132a) and purified sufficiently to separate it from the trimethylamine dehydrogenase activity also present. OXIDATION OF METHYLAMINE

Two mechanisms for methylamine ox idation occur among methylotrophic bacteria, one a direct route and one involving N-methylglutamate as an intermediate. The direct route is cat alyzed by methylamine dehydrogenase, which used phenazine methosulfate (PMS) as electron acceptor in vitro and catalyzes the following reaction: CH3NH2+ + PMS + H20 � HCHO + NH4+ + PMSH2. The enzyme was first described in Pseudomonas AMI (68) and subsequently was found in Pseudomonas WI (48), bacteria C2Al and W3Al (40, 43), Methylomonas methylovora (131), Pseudomonas sp. J. (125), and Pseudomonas oleovorans, and Mycobacterium sp. 10 (123). The second indirect route for methylamine oxidation involves two en zymes, N-methylglutamate synthase (EC 2.1.1.21) and N-methylglutamate dehydrogenase (EC 1.5.99.5), . which catalyze reactions 9 and 10, respectively. COOH · CHz · CHz · CH(NHz)· COOH + CH3NHz � COOH ' CH2' CHz ' CH(NHCH3) ' COOH + NH3•

9.

COOH · CH2 · CHz ·CH(NHCH3)COOH + X + H20 � XH2 + HCHO + COOH · CH2 · CH2 ·CH(NH2)· COOH.

10.

N-methylglutamate synthase was first described in Pseudomonas MA (180) and subsequently was purified by Pollock & Hersh (154). N-methyl glutamate dehydrogenase activity was then discovered in particulate frac tions of the same microorganism (86) and the two enzymes were recognized as a potential indirect route for methylamine oxidation. N-methylglutamate dehydrogenase has been solubilized and partially purified from Pseudomo nas MA (87) and was completely purified from Pseudomonas aminovorans (13, 14). Both enzymes use 2,6-dichlorophenol-indophenol as in vitro elec tron acceptor. The enzyme from P. aminovorans has a molecular weight of about 550,000 and is probably a tetramer (14). In contrast, the same enzyme from Pseudomonas methylica is both soluble and NAD linked (139), and an NAD-linked N-methylglutamate dehydrogenase is also found in extracts of Hyphomicrobium vulgare ZN (120). The enzyme from Hyphomicrobium

C1 METABOLISM

505


X, however, resembles those from P. aminovorans and Pseudomonas MA ( 132). Early work on the assimilation of labeled methylamine by Pseudomonas MS suggested the involvement in methylamine metabolism of two other N-methyl amino acid derivatives, N-methylalanine and l'-glutamyl me thylamide (105, 106). N-methylalanine is synthesized by N-methylalanine dehydrogenase ( 1 1 7), which catalyzes the reaction CH3 ' CO ' COOH + NADPH + H+ + CH3NH2 � -+ CH3CH (NHCH3) ' COOH + NADP+ + H20. N-methylalanine is an alternative substrate for N-methylglutamate dehy drogenase ( 1 3, 87), and these two enzymes could constitute another indirect mechanism for the oxidation of methylamine. However, N-methylalanine dehydrogenase, although present, apparently is not important during the growth of P. aminovorans on trimethylamine (15, 26). The comparatively low affinity of N-methylglutamate dehydrogenase for N-methylalanine ( 1 3, 87) also makes this route seem unattractive. l'-Glutamylmethylamide is the earliest detectable product of methyla mine metabolism in P. oleovorans, Pseudomonas MA, and Pseudomonas MS (105, 1 1 5, 1 1 9). It is synthesized by l'-glutamylmethylamide synthetase (EC 6.3.4. 1 2), 2 Mn + COOH · CH2 · CH2 · CH (NH2) ' COOH + CH3NH24 ATP CO(NHCH3) · CH2 ·CH2 · CH(NH2) · COOH + ADP + Pi ,

-

1 1.

which apparently is distinct from glutamine synthetase ( 1 1 5). The enzyme is also present in Hyphomicrobium ZV (1 20). Hyphomicrobium X (1 32). and P. aminovorans ( 1 5). Extracts of Hyphomicrobium ZV apparently also contain an enzyme the converts y-glutamylmethylamide to N-methyl glutamate, and it is possible that this enzyme, together with l'-glutamylme thylamide synthetase and N-methylglutamate dehydrogenase. constitutes a route for the oxidation of methylamine in some methylotrophs that do not possess methylamine dehydrogenase. Many yeasts can utilize methylamine as nitrogen source although none can grow on it as sole carbon and energy source (57a). Methylamine is oxidized to formaldehyde and ammonia by a monoamine oxidase that is present with catalase in peroxisomes. Formaldehyde and formate dehy drogenases are also induced allowing the provision of NADH and hence ATP from the oxidation of methylamine. Some of these yeasts can utilize methanol as carbon and energy source and hence can synthesize the en zymes necessary to assimilate formaldehyde into cellular material. Their inability to grow on methylamine is therefore inexplicable at the present time.

506


Oxidation of Formaldehyde Among C1-utilizing microorganisms the oxidation of formaldehyde occurs via at least two different routes. The direct route, which has formate as an intermediate, involves two enzymes, formaldehyde dehydrogenase and for mate dehydrogenase (reactions 12 + 13).


HCHO + X + H20 HCOOH + NAD+

�

�

12.

HCOOH + XH2•

13.

CO2 + NADH + H+.

A modification involving S-hydroxymethylglutathione and S-formyl glutathione as intermediates occurs in methanol-utilizing yeasts (56). Al though all C\-utilizing microorganisms apparently oxidize formate by an NAD-dependent formate dehydrogenase (EC 1.2. 1.2), several different types of formaldehyde dehydrogenase are found. The reader is referred to Stirling & Dalton (197) for a description of the bacterial formaldehyde dehydrogenases and to Kato, Sahm & Wagner (96) for similar information concerning methylotrophic yeasts. There is now considerable evidence for the presence of a cyclic mecha nism for formaldehyde oxidation in many C1-utilizing microorganisms that use the RMP pathway of formaldehyde oxidation. This dissimilatory RMP pathway is shown in Figure 6. High activities of the enzymes of the cycle have been found in several methyl amine- and methanol-utilizing bacteria (18, 21, 43, 171, 186, 200). The dissimilatory RMP cycle has also been implicated in formaldehyde oxidation in methane-oxidizing bacteria (199) and in methanOl-utilizing yeasts (95). In the latter case, however, the spe cific activities of glucose-6-phosphate dehydrogenase and 6-phophogluconHCHO

�

U6P

Ru5P

�

F6P

J

G6P

4

6P G Figure 6

LNAOlPt

� �

lP) + H +

Cyclic mechanism for the oxidation o f formaldehyde to CO2 (dissimilatory RMP).

3, glucose phosphate isomerase; 4, glucose-6-phosphate dehydrogenase; 5, 6-phosphogluconate

dehydrogenase. Otherwise the abbreviations are given in Figure 1.


c) METABOLISM

507

ate dehydrogenase are lower during growth on methanol than 9uring growth on non-C, compounds. A different cyclic pathway for formaldehyde oxidation has been formu lated for Pseudomonas MA, a methylamine-utilizing bacterium that assimi lates C, compounds via the serine pathway (140). This rather complex mechanism involves the synthesis of acetyl-CoA from formaldehyde and CO2 by the serine pathway (see Figure 3), followed by oxidation of the acetyl-CoA by the TCA cycle. No direct evidence supports the operation of this cycle. Indirect support comes from the observation that the PEP carboxylase (BC 4. 1 . 1 .31) of Pseudomonas MA is allosterically regulated by NADH and ADP (135, 140), as might be expected for an enzyme located at the branching point between the assimilatory serine pathway and its proposed dissimilatory counterpart (140). ENZYMOLOGY OF Cl OXIDATIONS

The 9 years since the appearance in this series of the last review on microbial C, metabolism (164) have seen major advances in the characterization of certain enzymes involved in C, oxidations. In our own and other laborato ries, interest has been centered on bacterial methane monooxygenase be cause of the importance of methane-oxidizing bacteria as potential sources of single-cell protein. As a result earlier problems associated with the prepa ration of active bacterial extracts have been overcome. Preliminary reports on the properties of the enzyme system from three methane-oxidizing bac teria have been published, and the methane monooxygenase from one of those strains, Methylococcus capsulatus (Bath), has been characterized in some detail. Trimethylamine dehydrogenase is another enzyme that has been the subject of detailed examination, this time by Steenkamp and his colleagues (1 89-1 94), and we now have a fair understanding of how this enzyme functions at the molecular level. We therefore thought it appropri ate to conclude this review with a section devoted to these enzymes and to others that have not yet been studied in as much detail, but about which our knowledge is rapidly advancing.

Methane Monooxygenase Since the review by Ribbons et al ( 1 64) methane oxidation in cell-free systems has been demonstrated in extracts from a number of methane oxidizing bacteria. The first report ( 1 65) showed that methane gave a two fold stimulation of NADH oxidation by a membrane preparation from cell extracts of Methylococcu$ capsulatus. NADH and oxygen were consumed in equimolar amounts, indicating a monooxygenase type reaction, i.e. CH4 + NADH + H+ + O2 � CH30H + H20 + NAD+, although a stoichiometric disappearance of methane or appearance of methanol was


508


not reported. The same stoichiometric relationship between NADH and O2 was later observed by Ferenci (70) with Methanomonas methanica particulate extracts. Ferenci also presented evidence that carbon monoxide was oxidized by the monooxygenase, confirming the earlier report (93) that whole cells of Methylosinus trichosporium (OB3b) and Methylomonas albus would oxidize CO to CO2, The methane monooxygenase assay methods to date had been indirect in that methane- or CO-stimulated disappearance of NADH or O2 were mea sured. In none of the assays was methane disappearance or methanol accu mulation followed. Our group at Warwick found that a soluble derivative of methane, bromomethane, could be used as an assay substrate for the enzyme by measuring its disappearance in the presence of NADH2 and O2 (36). The first report of a partial purification of the enzyme came from the Kent group, who made use of an earlier observation (88) that high concen trations of phosphate inhibited methanol oxidation in some methane oxidiz ers. Armed with this they followed methanol accumulation in the presence of 1 50 mM phosphate in cell extracts of M trichosporium OB3b (206). All methane-oxidizing activity was located in particulate fractions that were sedimented at 1 50,000 X g for 90 min, and both NADH and ascorbate were equally effective electron donors. The authors discounted the possibility that ascorbate was reducing endogenous NAD+ by some form of reversed electron flow by showing that amytal completely inhibited NADH-depend ent methane oxidation but had no effect on ascorbate-dependent methane oxidation. They concluded that ascorbate was probably directly reducing a CO-binding cytochrome c, which was essential for monooxygenase activ ity, but that electrons were passed from NADH along an electron transport chain to the physiological donor. In the following year Colby & Dalton (33) reported that a soluble me thane-oxidizing system could be reproducibly extracted from M capsulatus strain Bath. In sharp contrast to the results obtained above by the Kent group they found that NADH only would act as an electron donor and ascorbate would not support activity. Inhibitor studies suggested that an electron transport chain was not involved in the passage of electrons from NADH to the soluble monooxygenase. Resolution of the soluble monooxygenase into three components could be achieved by ion exchange and molecular sieve chromatographic tech niques (34). All three components were unstable above O°C. Fraction A was reasonably stable at O°C, but fractions B and C were very unstable unless kept in the presence of phenylmethylsulfonylfluoride and sodium thio glycollate, respectively. However, all fractions were stable for at least 3 months if kept below -20°C. Fraction A has a molecular weight of about 200,000 and subunits of molecular weight 68,000 and 47,000. The protein


C1 METABOLISM

509

contains 2 g atoms of iron and has an electron paramagnetic resonance (EPR) signal upon reduction with sodium dithionite, which is considerably enhanced in the presence of the substrate ethylene, suggesting that protein A binds substrate only when the protein has been reduced. The reductant for the complex, NADH, will not reduce protein A directly but reduces the protein C (34). This protein has a molecular weight of 44,600 and contains 1 mol of FAD, 2 g atoms of non-heme iron, and 2 mol of acid-labile sulfide per mol of protein (35). Core extraction and EPR studies suggest that the protein is of the [2 Fe-2S· (S-CYS)4] class of non-heme iron proteins. The appearance of a semiquinone upon reduction with NADH may represent a semireduced intermediate that could be an important component in the electron transport pathway to the monooxygenase. The other component, protein B, is colorless and has a molecular weight of about 15,000. At present we are uncertain of its role in the reaction, The properties of the component proteins from M. capsulatus appear to be quite different to those reported from M trichosporium OB3b. The three components from the latter organism have been purified. They comprise a soluble CO-binding cytochrome c of molecular weight 1 3,000, a colorless copper-containing protein of molecular weight 47,000, and an uncharacter ized protein of molecular weight 9,400 (205). The purified components were stable on storage at O°C, but they lost activity on freezing. In its purified form the enzyme system does not use NAD(P)H as electron donor; only ascorbate will so serve by directly reducing the CO-binding cytochrome c. Reducing power for the purified monooxygenase components could also be furnished by a partially purified methanol dehydrogenase preparation and methanol-a non-NADH-linked reaction. The authors noted that an elec tron recycling scheme independent of NADH would be consistent with observed cell-yield data if insufficient NADH was generated from subse quent reactions to supply the monooxygenase. However, recent evidence from our laboratory does not support either the electron recycling concept or its raison d'etre, the supposed insufficient supply of NADH. Using the OB3b strain we have observed that the only effective electron donor for the system is NAD(P)H (196) and that neither ascorbate nor methanol will serve as electron donor. Furthermore, we have good evidence that an NAD-linked formaldehyde dehydrogenase is in this strain, which coupled with the NAD-linked formate dehydrogenase could provide reducing power for the monooxygenase as NADH, although cell yields might be less than if an electron recycling scheme were operative. In our hands the OB3b methane monooxygenase system is soluble, stable to freezing, and insensitive to amytal and cyanide concentrations below ImM. These results are quite different to those reported by Tonge et al (205, 206) and are in close agreement with those reported by Colby & Dalton (33-35) for the M. capsulatus (Bath) enzyme. Stirling & Dalton (198) also observed


5 10


that cross-reactivity occurred between components B + C from M. cap sulatus and one of the components from the OB3b strain, suggesting a close functional relationship between the two enzyme systems. In addition to methane and carbon monoxide a wide variety of other substrates have been observed to be oxygenated by the monoxygenase com plex from M capsulatus (Bath), M. trichosporium (OB3b), and Me thylomonas methanica (37, 196). They include alkanes, alkenes, dimethyl and diethyl-ether, alicyclic, aromatic, and heterocyclic compounds, and ammonia (49). Qualitatively, the range of oxidations by the Bath and OB3b strains were similar; the system from M. methanica, however, was more restricted in that it did not oxidize aromatic, alicyclic, or heterocyclic compounds (196).

Methanol Dehydrogenase (EG 1. 1. 99. 8) Methanol dehydrogenase (EC 1 . 1 .99.8) apparently occurs in all methanol utilizing bacteria, although not in yeasts, and has been purified from 1 6 strains. I t catalyzes the reactions R CH20H + PMS .... R CHO + PMS H2 and HCHO + H20 + PMS .... HCOOH + PMS H2, where R is hydrogen or an alkyl group and PMS is phenazine methosulfate. Formalde hyde (and usually acetaldehyde) are oxidized because they occur as the hydrated form in aqueous solution (185). The enzyme from all sources has a high pH optimum for activity (PH 9-1 1) and when purified requires ammonia or primary amines for activity. The enzymes can be roughly divided into four groups according to their properties. Group 1 , enzymes from Pseudomonas M27 (5), Methylococcus capsulatus (Texas) (145), Hy phomicrobium WC ( 1 85), Pseudomonas TPI (1 85), Pseudomonas W I ( 1 85), Pseudomonas 249 1 (223), Hyphomicrobium X (64), Pseudomonas fluorescens S25 and S50 (224), Pseudomonas RJ l (129), and Paracoccus denitrificans (44), catalyze the oxidation of primary alcohols only, require ammonia or methylamine for activity, and are dimeric proteins of molecular weight about 120,000 ( 1 50,000 for the enzymes from P. denitrificans and P. fluorescens S50). Enzymes within the group differ in their isoelectric points and amino acid compositions. Group 2 are those enzymes from Methylobacterium organophilum (219) and Pseudomonas C (79); they re semble those from group 1 except that they also oxidize some secondary alcohols. Group 3 enzymes are from Methylomonas methanica (148) and Methylosinus sporium ( 146) and are monomeric with molecular weights of 60,000. Otherwise they resemble the group 1 enzymes. Group 4 is com prised of Rhodopseudomonas acidophila which contains an alcohol dehy drogenase that differs from the group 1 enzymes in its ability to oxidize secondary alcohols, in its very high apparent Km for methanol (but not for CZ-CiO primary alcohols), in its activation by higher aliphatic amines in preference to ammonia, and in its sensitivity to oxygen (17). Bamforth &


C1 METABOLISM

511

Quayle (17) suggest that this enzyme is evolutionary unrelated to, or on the other hand represents a primitive version of, the methanol dehydrogenase found in other C 1 -utilizing bacteria. Until recently, little was known about the mechanism of action of me thanol dehydrogenase. Anthony & Zatman (6) observed that the prosthetic group resembled a pteridine in its spectral properties and a lumazine deriva tive was suggested ( 1 85). From recent work in Holland, however, it is now clear that the enzyme is in the reduced form when isolated (63), the spectral properties of the oxidized enzyme being very different with an absorption maximum at 400-4 1 0 nm and no maximum at 350 nm. EPR spectra indicate that the prosthetic group is a quinone containing two nitrogen atoms (63, 64, 214a) and therefore is inconsistent with a pteridine prosthetic group; in addition, evidence for an intermediate semiquinone form of the prosthetic group has been obtained. These Dutch workers have also established that ammonia and high pH are required only for the reoxidation of the reduced enzyme, whereas the initial reaction of the oxidized enzyme with substrate occurs readily at pH 7 in the absence of ammonia.

Methylamine Dehydrogenase The enzyme methylamine dehydrogenase purified to homogeneity from Pseudomonas AM I (68, 1 8 1), Pseudomonas J (1 25), and Methylomonas methylovora (13 1), catalyzes the oxidation of primary amines and diamines to the corresponding aldehydes using phenazine methosulfate as electron acceptor. The natural electron acceptor may be a cytochrome c-5 5 1 (2, 125). Kinetic analysis (69) suggests a ping pong mechanism for the enzyme (Figure 7). The enzymes from Pseudomonas AM 1 and Pseudomonas J have been extensively studied (69, 1 26, 127). They are of similar molecular weight, ca 1 05,000, do not contain iron or copper, have absorption maxima at 278 nm, 330 nm, 430 nm, and 460 nm (shoulder), and fluorescence maxima at 330 nm (tryptophan) and 380 nm. Unfortunately the early suggestion (69) that the prosthetic group is a pyridoxal derivative has neither been confirmed nor refuted. However, considerable progress has been made by Matsumoto et al (125-128) on the quaternary structure of the enzyme. It contains two types of subunits, light (molecular weight

E

r

C H3NH2 Figure 7

I

HCHO

E'

r

PMS

I

E

PMSH2+NH3

Proposed ping pong kinetics of methylamine dehydrogenase.


512


13,(00) and heavy (molecular weight 40,(00) in a a2/32 configuration. The light subunit carries the covalently bound chromophore, although its spec tral characteristics are modified. Mixing the heavy and light subunits to gether restores enzyme activity, the original spectrum, and a free-radical EPR signal that is observed with native enzyme but not with the isolated subunits. The amino acid composition of both subunits from the Pseudomo nas AMI and Pseudomonas J enzymes have been determined. The light subunits are very similar, but there are considerable differences in the heavy subunits and these are thought to cause the differences in heat stability and isoelectric point exhibited by the two native enzymes. Recent studies (128) using the cross-linking reageant dimethylsuberimidate have confirmed the a2f32 arrangement of the subunits and suggest that the enzyme is assembled around a core consisting of two heavy subunits.

Trimethylamine dehydrogenase (EG 1. 5. 99. 7)

Trimethylamine dehydrogenase purified from bacteria 4B6 and W3Al cata lyzes the oxidative N-dealkylation of trimethylamine, diethylamine, and a very few other tertiary methyl- and ethyl amines using phenazine methosul fate as in vitro electron acceptor (4 1 , 1 9 1). The natural electron acceptor is now known to be a flavoprotein (1 89). The kinetics of diethylamine oxidation suggest a ping pong mechanism (191) (Figure 8). The molecular weight of the enzyme is 146-161 X 103 and it contains non-identical subu nits of molecular weight 70-80 X 103 (38, 4 1 , 1 9 1). An unusual property of trimethylamine dehydrogenase is its sensitivity to monoamine oxidase inhibitors of the substituted hydrazine and non-hydrazine types. (41). Steenkamp and his colleages ( 1 89-1 94) have subjected trimethylamine dehydrogenase to detailed scrutiny by using a variety of sophisticated physi cal and chemical techniques, including absorbance, Buorescence, EPR, and NMR spectroscopy, core extrusion, amino acid sequencing, stopped-Bow spectrophotometry, and rapid-freeze EPR. These have shown the enzyme to contain two types of prosthetic groups, a covalently bound organic chromophore initially thought to be pteridine ( 1 93) but now known to be an unusual 6-substituted FMN derivative, probably 6-S-cysteinylriboBavin5'-phosphate (190, 1 92), and an [Fe4S 4(S-Cys)4] core unit (89, 1 94). The

B

A

1[

EB Figure 8

1

1[

1

FA

Proposed ping pong kinetics of trimethylamine dehydrogenase. A, diethylamine;

B, phenazine methosulfate; Po ethylamine; Q, reduced phenazine methosulfate; R. acetalde hyde.

C1 METABOLISM

513


flavin is rapidly reduced by substrate, but EPR studies have demonstrated a much slower substrate-dependent interaction between reduced flavin and the Pe-S center, resulting in the formation of a spin-coupled complex of flavin semiquinone and reduced Pe-S center (194). The kinetics of this interaction indicate that it is the rate-limiting step, being much slower than either the initial reduction of the flavin or the reoxidation of the enzyme with phenazine methosulfate. Literature Cited 1 . Andreev, L. V., Trotsenko, Yu. A., Gal chenko, V. P. 1977. In Microbial Growth on CrCompounds, ed. G. K. Skryabin, M. V. Ivanov, E. N. Kondrat jeva, G. A. Zavarzin, Yu. A. Trotsenko, A. 1. Nesterov, pp. 15-17. USSR Acad. Sci.: Puschino 2. Anthony, C. 1975. Sci Prog. 62:1 67206 3. Anthony, C. 1978. J. Gen. MicrobioL 104:91-104 4. Anthony, C., Zatman, L. J. 1964. Rio chem. J. 92:609-14 5. Anthony, C., Zatman, L. J. 1965. Rio chem. J. 96:808-12 6. Anthony, C., Zatman, L. J. 1967. Rio chem. J. 104:9�9 7. Attwood, M. M., Harder, W. 1973. An tonie van Leeuwenhoek J. MicrobioL Serol 39:357 8. Attwood, M. M., Harder, W. 1974. J. Gen. Microbiol 84:350-56 9. Attwood, M. M., Harder, W. 1977. FEMS Lett. 1 :25-30 10. Attwood, M. M., Harder, W. 1978. FEMS Lett. 3:1 1 1-14 1 1 . Babel, W., Hofmann, K. 1975. Z. AI/g. MikrobioL 15:53-57 12. Babel, W., Miethe, D. 1974. Z AI/g. MikrobioL 14:1 53-56 13. Bamforth, C. W., Large, P. J. 1977. Rio chem. J. 161 :357-71 14. Bamforth, C. W., Large, P. J. 1977. Rio chem. J. 167:509-12 1 5 . Bamforth, C. W., O'Connor, M. L. 1979. J. Gen. Microbiol 1 10: 143-49 16. Bamforth, C. W., Quayle, J. R. 1977. J. Gen. Microbiol 101 :259-67 17. Bamforth, C. W., Quayle, J. R. 1978. Riochem. J. 169:677-86 18. Beardsmore, A. J., Quayle, J. R., Tay lor, I. J. 1978. Proc. Soc. Gen. MicrobioL 5:41-42 19. Bellion, E., Spain, J. C. 1976. Can. J. Microbiol. 22:404-8 20. BeIlion, E., Woodson, J. 1975. J. Bac terial 122:557-64 2 1 . Ben-Bassat, A., Goldberg, I. 1977. Bio chim. Biophys. Acta 497:586-97

22. Bird, C. W., Lynch, C. M., Pirr, P. J., Reid, W. W., Brooks, C. J. W., Mid dleditch, B. C. 197 1 . Nature 230:473 23. Blackmore, M. A., Quayle, J. R. 1968. Biochem. J. 107:705-1 3 24. Blackmore, M. A., Quayle, J . R. 1970. Riochem. J. 1 1 8:53-59 25. Boulton, C. A., Crabbe, 1. C., Large, P. J. 1974. Riochem. J. 140:253-63 26. Boulton, C. A., Large, P. 1. 1977. J. Gen. MicrobioL 101 : 1 5 1-56 27. Brook, D. P., Large, P. 1. 1975. Eur. J. Biochem. 55:601-9 28. Brook, D. P., Large, P. J. 1976. Bio chem. J. 157:197-206 29. Bykovskaya, S. V., Luchin, S. V. 1977. In Microbial Growth on CrCompounds, ed. G. K. Skryabin, M. V. Ivanov, E. N. Kondratjeva, G. A. Zavarzin, Yu. A. Trotsenko, A. I. Nesterov, pp. 62-63. USSR Acad. Sci.: Puschino 30. Chandra, T. S., Shethna, Y. I. 1976. Curro Sci. 45:653-58 3 1 . Chandra, T. S., Shethna, Y. I. 1977. J. BacterioL 1 3 1 :389-98 32. Chernyadiev, I. I., Terekhova, I. V., Doman, N. G. 1977. In Microbial Growth on CrCompounds, ed. G. K. Skryabin, M. V. Ivanov, E. N. Kondrat jeva, G. A. Zavarzin, Yu. A. Trotsenko, A. I. Nesterov, p. 132. USSR Acad. Sci.: Puschino 33. Colby, J., Dalton, H. 1976. Biochem. J. 1 57:495-97 34. Colby, J., Dalton, H. 1978. Biochem. J. 1 7 1 :461-68 35. Colby, J., Dalton, H. 1979. Biochem. J. 177:903-8 36. Colby, J., Dalton, H. Whittenbury, R. 1975. Riochem. J. 1 5 1 :459-62 37. Colby, J., Stirling, D. I., Dalton, H. 1977. Biochem. J. 165:395-402 38. Colby, 1., Zatman, L. 1. 197 1 . Biochem. J. 1 2 1 :9P 39. Colby, J., Zatman, L. J. 1972. Biochem. J. 128:1 373-76 40. Colby, I., Zatman, L. 1. 1973. Biochem. J. 1 32:101-12


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64. Duine, J. A., Frank. J .• Westerling, J. 1978. Biochim. Biophys. Acta 524: 277-87 65. Dunstan, P. M., Anthony, C., Drabble, W. T. 1972. Biochem. J. 1 28:99-106 66. Dunstan, P. M., Anthony. C., Drabble, W. T. 1972. Biochem. J. 128: 107-15 67. Eady, R. R.• Jarman, T. R., Large, P. J. 1971. Biachem. J. 125:449-59 68. Eady, R. R.• Large, P. J. 1968. Biochem. J. 106:245-55 69. Eady, R. R., Large, P. J. 1971. Biochem. J. 123:757-71 70. Ferenci. T. 1974. FEBS Lett. 41 :94-98 7 1 . Ferenci. T., Strom, T., Quayle, J. R. 1974. Biochem. J. 144:477-86 72. Ferenci, T.• Strom, T., Quayle, J. R. 1975. J. Gen. Microbiol 91 :79-9 1 73. Fuchs, G., Andress, G., Thauer, R. K. 1976. In Microbial Production and Uti lization o/Gases, ed. H. G. Schlegel. G. Gottschalk, N. Pfennig, pp. 23 1-36. Gottingen: Goltze 74. Fuchs, G., Schnitker, U.• Thauer, R. K. 1974. Eur. J. Biochem. 49: 1 1 1-15 75. Fujii, T., Tonomura, K. 1973. Agric. Biol Chem. 37:447-49 76. Fujii, T., Tonomura, K. 1974. Agric. Diol Chem. 38:1 763-65 77. Fukui. S., Tanaka, A., Kawamoto, S., Yusahara. S., Tanaka, A.• Osumi, M., Imaizumi, F. 1975. Eur. J. Biochem. 59:561-66 78. Fukui. S.• Tanaka, A .• Kawamoto, S., Yusahara, S., Terashini, Y., Osumi, M. 1975. J. Bacterial. 123:317-28 79. Goldberg, I. 1 976. Eur. J. Biochem. 63:233-40 80. Goldberg. I., Mateles, R. I. 1975. J. Bacterial. 124:1028-29 8 1 . Hampton, D. 1974. Bacterial catabol ism of tetramethylammonium chloride. PhD thesis. Univ. Reading, Reading, U.K. 264 pp. 82. Hampton, D., Zatman, L. J. 1973. Bio chem. Soc. Trans. 1 :667-68 83. Hazeu, W.• Batenburg-van den Vegte, W. H., Nieuwdorp, P. J. 1975. Ex perientia 3 1 :926-27 84. Hersh, L. B. 1973. J. Bial Chem. 248:7295-303 85. Hersh, L. B., Bellion, E. 1972. Biochem. Biophys. Res. Commun. 48:7 1 2-19 86. Hersh. L. B .• Peterson. J. A., Thomp son, A. A. 1971. Arch. Diochem. Bia phys. 145: 1 1 5-20 87. Hersh, L. B., Stark. M. J., Worthen, S., Fiero, M. K. 1972. Arch. Biachem. Bio phys. 1 50:219-26 88. Higgins, I. J., Quayle, J. R. 1970. Bia chem. J. 1 1 8:21(}'-18


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