N-(carboxyalkyl)amino acids: occurrence, synthesis, and functions.

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Annu. Rev. Biochem. 1992.61:517-554. Downloaded from www.annualreviews.org by Yale University - SOCIAL SCIENCE LIBRARY on 10/02/13. For personal use only.

Annu. Rev. Biochem. 1992. 61:517-57

N-(CARBOXYALKYL)AMINO ACIDS: ! Occurrence, Synthesis, and Functions John Thompson and Jacob A. Donkersloot Laboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892

KEY WORDS:

opines, pyruvate:oxidoreductase, crown gall, NAD(P)-binding domains, amino acid dehydrogenase

CONTENTS PERSPECTIVES AND SUMMARy .......... . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . NOMENCLATURE AND PROPERTIES OF N-(CARBOXYALKYL)AMINO ACIDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . CHEMICAL AND ENZYMATIC SYNTHESES OF N·(CARBOXYALKYL)AMINO ACIDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . N-(CARBOXYALKYL)AMINO ACIDS IN BACTERIA . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .... . . . N·(Carboxyalkyl)Amino Acids in Lactococcus lactis . . . . . . . . . . . . . . . . . . . ... . . . . . . .... . . . . . . .

Biosynthesis of N 5.(Carboxyethyl)ornithine and JV6-(Carboxyethyl)lysine... . . . . . . . ... . N5-(l·L·Carboxyethyl)·L-ornithine:NADP + Oxidoreductase . . . . . . . . . . . . . . . . . . .. ........... N5-(C,a!'boxyeth )ornithine Syntha�e: . Genetic Locus and Linkage. . . . . . . . . . . . . . . . . . . . . . InhIbit ion of N -(Ca rboxyethyl)ormthme Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N2_(I-D-Carboxyethyl)'L-Phenylalanine Dehydrogenase in Arthrobacter. . . . . . . . . . . . . . .

i

N-(CARBOXYALKYL)AMINO ACIDS IN PLANTS (CROWN GALL) .... . . . . . . ... . . . . Octopine and Nopaline Families. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biosynthesis of Crown Gall Opines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-Octopine Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-Nopaline Synthase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . Functions of Opines in Crown Gall. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catabolism of Opines by Agrobacterium tumefaciens .....................................

N-(CARBOXYALKYL)AMINO ACIDS IN MARINE INVERTEBRATES . . . . . . . . . . . . . .

Biosynthesis

of Opines

in Marine Invertebrates............................................

N-(CARBOXYALKYL)AMINO ACIDS IN YEASTS . . . . .. . . . .. . . . . . . . ....... ... .. . . . . . . . . . .

518 519 519 522 523 523 524 525 526 527 528 528 528 531 531 532 533 535 535 537

IThe US Government has the right to retain a nonexclusive royalty-free license in and to any copyright covering this paper

517

518

N-(CARBOXYALKYL)AMINO ACIDS: OTHER SOURCES...............................

538

N-(CARBOXYALKYL) DlPEPTIDES AS ENZYME INHIBITORS.......................

539

N-(CARBOXYALKYL) DlPEPTIDES IN ENZYME PURIFICATION....................

541

SEQUENCE ANALYSIS OF N-(CARBOXYALKYL)AMINO ACID DEHYDROGENASES ... ..... ... . ... .... . .. ..... . ... ..... . . ..... ... . . ...... . .. . ..... . . . Nopaline and Octopine Synthases .. ...... ...... . ....... . . . . . . .... . . . . . ... . . . . . . .... .. . . . .... . . Saccharopine Dehydrogenase. ............. . . .... . . ............ . . . ... . . ................ . . .... . . . 5-(Car oxyethyl)ornithine Synthase .......... .. . . . . ...... . . .... . .. . . . . .... . .. . . . .... ......... Global Srmliarltles .. ......... ...................... .............. . . ............ ........ ..... . . Dinucleotide-Binding Domains .... ...... .. . . . ..... . ....... . . . . . ................ ......... . . . . ...

543 543 543 544

'f


THOMPSON & DONKERSLOOT

�

544

545

REACTION MECHANISM(S) AND CATALYTIC DOMAINS ..... . . ....... . . . ....... . . ..

549

CONCLUSIONS AND FUTURE DIRECTIONS ...............................................

550

PERSPECTIVES AND SUMMARY The properties and function of the constituent amino acids of proteins have been presented in classic texts by Greenstein & Winitz (1), Meister (2), and in more recent publications by Bender (3) and Barrett (4) . In their treatise of 1961, Greenstein & Winitz described 90 or so amino acids, but in 1985, Hunt (5) tabulated approximately 700 amino and imino acid derivatives. Many of these "new" compounds either are derivatives of the 20 "primary" amino acids, or originate by posttranslational modifications of these residues in proteins. In a comprehensive review, Wold (6) mentions 40 such derivatives of lysine alone! An unusual modification occurs when the a- or UJ-NH2 group of an amino acid undergoes reductive condensation with the ketone carbonyl of an a-keto acid. The products of these reactions, collectively termed N-(carboxy alkyl)amino acids [N-(CA)amino acids] , have been isolated from phylogene tically diverse sources including eukaryotic cells, plant tumors, and muscle tissue of marine invertebrates (Table 1) . N-(CA)amino acids have also been identified during studies of certain enzyme-catalyzed reactions. In the phar maceutical industry, dipeptides containing N"'-(CA)-moieties have been syn thesized for therapeutic use. N-(CA)amino acids have not previously been discussed in Annual Review of Biochemistry. The invitation from the Editorial Committee to summarize biochemical aspects of these compounds stems in part from our discovery of N"'-(carboxyethyl)amino acids in bacteria [Lactococcus lactis (7-9; Table 1)]. More importantly, the request provides the opportunity to overview the N-(CA)amino acids as a group, and to highlight the different roles these compounds play in the physiologies of very diverse organisms. Additionally, we reflect on how knowledge gained from the study of these compounds has contributed to developments in drug design, and to the genetic engineering of plants. Finally, we speculate on future developments in this exciting area of amino acid biochemistry.

N-(CARBOXYALKYL)AMINO ACIDS

5 19


NOMENCLATURE AND PROPERTIES OF N-(CARBOXYALKYL)AMINO ACIDS The general structures of the N"'- and NW-(CA)amino acids are shown in Figure I. These compounds may be regarded as two amino acids linked via a common imino nitrogen or as secondary amines. From a biosynthetic view point, the N-(CA)amino acids [which are frequently called opines (10)] are the products of the NAD(P)H-dependent reductive condensation between an a-keto acid and the a- or w-NH2 group of an amino acid (11) . In this review, the terms synthase and dehydrogenase are used interchangeably with refer ence to the NADCP) +-dependent oxidoreductases responsible for the biosynthesis of N-(CA)amino acids (Table I). These amino acids usually have two asymmetric centers, and in nature may exhibit (L,L) or (D,L) stereochem istry. However, when synthesized chemically, they are often a mixture of these diastereomeric forms. The nomenclature for the N"'- or NW-compounds is provided by the examples shown in Figure 1. Thus for A and B (n=4),when 2 R=RJ =CH3 the N-(CA)amino acids are N -11 -(DL)-carboxyethyl]-L-alanine and N6-[I-(0L)-carboxyethyl]-L-lysine, respectively. Many N-(CA)amino acids possess both primary and secondary amine groups and the molecules contain two or more carboxyl groups. Although amphoteric, the N (CA)amino acids are less basic than the parent amino acids, and derivatives of arginine, lysine, and ornithine exhibit neutral or even slightly acidic proper ties.

CHEMICAL AND ENZYMATIC SYNTHESES OF N-(CARBOXYALKYL)AMINO ACIDS Two excellent reviews (10, 1 2) provide details of the physical properties, chemical syntheses, and methods for extraction, detection, and structure determination of many N-(CA)amino acids. The Abderhalden-Haase pro cedure ( 1 3), in which free or N-protected amino acids are reacted with the resolved (0) or (L) enantiomers of a-halo acids under alkaline conditions, has frequently been employed for the stereoselective synthesis of N-(CA)amino acids (14-16). Thus the reaction between L-arginine and a-(L) bromopropionate yields N2-(1-0-carboxyethyl)-L-arginine, the naturally occurring isomer of octopine (17, 1 8) . Reductive condensation between an amino acid and the desired a-keto acid using NaBH4 or NaBH3CN also provides an efficient, but nonstereospecific method of synthesis (1 8-20). An excellent method for the diastereoselective synthesis of N"'- and N W_ (CA)amino acids has been reported (21), whereby the trifluoromethanesulfon ates of enantiomerically pure lactates, {3-phenyllactates and dimethyl a hydroxyglutarate, are reacted with suitably protected esters of (0)- or (L)-a-


Vl N o

Table 1

@ o

Naturally occurring N-(carboxyalkyl)amino acids: distribution and biosynthesis

N-(carboxyalkyl) amino acid

Trivial name

Enzyme

Cofactor

Do - ctopine synth.ase

NAD(P)H

M· r

Referencesb

Plants (crown gall) N2_( I-D-Carboxyethyl)-L-arginine

octopine

N2_( I-D-Carboxyethyl)-L-ornithine

octopinic acid

N2_( I-D-Carboxyethyl)-L-lysine

lysopine

N2_( ID - C - arboxyethyl)L - h - istidine

histopine

N2-( I-D-Carboxyethyl)-L-methionine

methiopine

(EC 1.5.1.11)

38,000

1 09

38,801c

110

38,7 33c

111

15 8,000 N2-(1,3-o-Dicarboxypropyl)-L-arginine

nopaline

N2_( 1, 3-o-Dicarboxypropyl)-L-ornithine

nopalinic acid

N2-( 1, 3-Dicarboxypropyl)-L-leucine

leucinopine

Dn - opaline synth.ase (EC 1.5.1.19)

(ornaline) N2-(1,3-Dicarboxypropyl)-L-asparagine

asparaginopine (succinamopine)

N2-(l,3-DicarboxypropYl)L - g - lutamine

glutaminopine cucumopine mikimopine

N2-(Carboxymethyl)-L-arginine

acetopine (noroctopine)

Enzyme s not yet characterized

NAD(P)H

114

(40,00 0)

114

(45,394)
-oE-- Amino Acids

I octopinic acid I Iysopine

histopine o ctopine

methiopine

CROWN GALL Figure 9

I

t

P yruvate

ornithine lysine

histidine arginine

methionine

I I I I LDH I I

+ .

laCtiC Acid

AD
-

.," 200

en

16 C. 0 Z

/

100

0

/

.,0> "

,/

2

,

200

Octopine Synthase

/

� 200 Q) c

.0. e '" .J:: " " '" en

/

100 ..

. /

100

/

-g. ..c:

/

,/ ' /

0

111 3OO '"

300

0

0

/

,

/

100

200

N5·(CE)Ornithine Synthase

300

Figure 16 Amino acid sequence comparison among the four N-(CA)amino acid de hydrogenases: NOS and OCS (left); SDH and CEOS (right) . The programs Compare (window size

=

20; stringency

compared (260).

=

1 1) and DotPlot were used (259). NOS and OCS have previously been


546


{3-strands of this fold are part of a four- or six-stranded parallel sheet, whose C-terminal edge is in close proximity to the pyrophosphate bridge of NAD(H) . Several amino acids play important roles in this interaction, and a "core fingerprint" for ADP-binding J3aJ3-folds has been proposed (262, 264, 265) . Within this fingerprint, the motif GXGXXG (single-letter amino acid code) is highly conserved (Figure 17). The first Gly residue marks the sharp turn from the first /3-strand (/3A) to the succeeding a-helix, and is located close to the pyrophosphate bridge. The second Gly, which is located at the N-terminus of the a-helix, permits a close approach between this terminus and the pyrophosphate moiety, which allows the charge on this group to be stabilized by the dipole originating from the helix (266) . The third Gly of the fingerprint facilitates a close interaction between the a-helix and the /3strands. In addition, a conserved acidic amino acid (Asp or Glu) was identi fied at the end of the second f3-strand (f3B) , which permits H-bonding between the carboxylate group of this amino acid and the 2 ' -hydroxy of the adenine ribose. Although less is known about NADPH-binding regions , they typically contain the GXGXXA motif (Figure 1 7 ; 265 , 267 , 268). The Gly-to-Ala substitution in the third position of the motif causes the {3a{3-fold to be slightly less compact (265). Furthermore, the end of the {3B strand of these enzymes does not usually terminate with an acidic residue, presumably to avoid the electrostatic repulsion that would occur with the ribose 2 ' phosphate (265 , 269) . Comparison of the sequences of several NADH- and NADPH-binding flavoprotein disulfide oxidoreductases indicates that in the latter group of enzymes two conserved Arg residues separated by five other amino acids may serve to neutralize the "extra" phosphate group of NADPH (268). Moreover, the three-dimensional structure of glutathione reductase shows that these residues are indeed close to this phosphate (270, 27 1 ) . Site-directed mutagenesis has confirmed the importance of these two Arg residues in the binding of NADPH by these oxidoreductases (268). In other NADP(H)-linked dehydrogenases (e.g. glutamate dehydrogenase, see Figure 17), amino acid residues located elsewhere in the sequence may serve the same function (272) . Recently,a new type of NADPH-binding domain was discovered in FAD containing ferredoxin-NADP+ reductases (273). This domain is characterized by a central five-stranded parallel f3-sheet and six surrounding helices. Whereas the actual NADPH-binding site is also located at the C-terminal edge of this ,B-sheet, the fingerprint of the pyrophosphate-binding loop appears to be MXXXGTGXXP. 5 N - (CARBOXYETHYL)ORNITHINE SYNTHASE

Purified CEOS has a strong preference for NADPH (38), and a motif near the center of the sequence closely resembles the core fingerprint for the binding of this cofactor (Figure


NADPCHI

Binding

N!5-ICEfOrnithine synthase IL lactis)

Glutamate dehydrogenase IE. com Glutattlione reductase (human) Mercuric reductase (5. aureus p1258)

'''�G S'G N V A a G A F S S 1 S K YIS S N �R K T M s 1 F K E N "'IG M R V S V SIG SIG N V A a Y A l E K A M E FIG AIR V

1 T

A siD S S G T V V D E S

",\G R S V 1 VIG A'G Y 1 A V E M A G 1 L S A LIG s@ LMI] R H D K V L R S F "'Ia R L A V 'IG S[GylliELGQ MFHN LIG TIE V T L M�R S E R L F K T Y D

Octopine syntflase

' MIA K V A I LIGAIG N V A L T l A G OLAR R lIG a[v s si-wAlp I S N R N S F N

Nopalil'1e synthase

" pIL T V G V LIG SIG H A G T A l A AW F A S RI . H v plT A L WAlp A D H P G S I S

(A. tumefaciensl

(A. tumefaciensl

ehydrogenase

Saccharopine d

IY. lipofvtica)

NADIHI

Binding

Lipoamide dehydrogenase . coli)

""'G A L G R e G s G A I 0 L A R K V G I P E E N

I I

RWD M N E T K K G G P F Q E

176 E �G GIG I I G L E M G T V Y H A LIG S�E M F D Q V I P A A D

IE. Phenvtalanine dehydrogenase

"'IG K T Y A I alG llG K V G Y K V A E Q L L K AIG AID L F V VJD I H E N V L N S I K

leucine dehydrogenase

miG K V V A V O)G V)G N V A Y H L e A H L H E EJG A�D I N K E V V A A A V

Alanine dehvcl,rogenase

1661G V H A R K V T V I [G GIG I A G T N A A K I A V G MIG A�D L 5 P E R l R Q L E

(B. sphaencus)

(B. stearothermophifus)

(B. sphaencus)

lactate dehydrogenase lB. steafDthermophiJus)

'IG A R V V V 'IG AIG F V G A S Y V F A L M N QIG ' IA 0 E I V L 1 1 0 A N E S K A I G

Alignment of putative nucleotide-binding folds of N-(CA)amino acid synthases and other dehydrogenases . The three strands that make up the putative f3af3 fold are boxed, and conserved amino acid residues are in bold type. The NADP-binding fold of glutathione reductase is derived from a three-dimensional model of the enzyme (271). The putative f3af3 folds of mercuric reductase and lipoamide dehydrogenase are based on the structural and sequence homologies of these disulfide oxidoreductases with glutathione reductase (268, 294). The prediction for glutamate dehydrogenase is based on earlier reports (268 , 276, 28 1 , 282). The octopine synthase and nopaline synthase predictions have been described previously (260). The alignment shown for the four Bacillus enzymes is slightly modified from an earlier prediction (295). References for the sequence data are: N5-(CE)omithine synthase (J. A. Donkersloot and 1. Thompson, unpublished information), glutamate dehydrogenase (274, 275), glutathione reductase (271) , mercuric reductase (296) , octopine synthase ( 1 10, 11 1 ), nopaline synthase (115, 1 1 6), saccharopine de hydrogenase (20 1 ) , Jipoamide dehydrogenase (297), phenylalanine dehydrogenase (298), leucine dehydrogenase (299), alanine dehydrogenase (295), lactate dehydrogenase (300) . Figure 1 7

? (3

�

� �

E �

z o

b �

VI .j:>. ...,J


548


1 7). Interestingly, 8 of the 9 amino acids in this segment are identical to the equivalent sequence of (NADPH-dependent) glutamate dehydrogenase (GDH) from E. coli (274, 275), Salmonella typhimurium (276) , yeast (277, 278) , and Neuro�pora crassa (279) . No structural data are available for CEOS, and until recently only a relatively low-resolution (0.6 nm) model (280) existed for GDH from Clostridium symbiosum. However, in 1 974, a J3aJ3-fold for nucleotide-binding was predicted for bovine liver GDH on the basis of sequence homologies to other ADP-binding units (28 1 ). Similar predictions have since been made for GDH from Neurospora, and Saccharo myces cerevisiae (277 , 282). The structure of the NAD+ -dependent GDH from Clostridium symbiosum was recently solved to 1 .96 A resolution. One of the two domains of this enzyme is indeed structurally similar to the classical dinucleotide-binding fold. Interestingly, however, the direction of the third strand of the (seven-stranded) f3-sheet is reversed (282a) . Based on these results and on the alignment shown in Figure 1 7 , a nucleotide-binding f3af3fold can be reasonably predicted for CEOS. When aligned in this fashion, Arg l 95 (or Lys196) and Lys202 of CEOS virtually coincide in relative position with the two Arg residues involved in the binding of NADPH by the flavoprotein disulfide oxidoreductases. If we assume that the size of the dinucleotide-binding domain in CEOS is similar to that of other dehydrogenases (about 1 30 residues) , then this domain would encompass, approximately, residues 1 60-290. One might envisage that by suitable folding of the polypeptide chain, amino acids from residue 290 to the COOH-terminus can interact with residues in the NHz-terminus, to form the catalytic domain of the enzyme. Monneuse & Rouze reported that the fingerprint for ADP binding is located close to the NHz-terminus of both proteins, and on this basis a {3a{3 nucleotide-binding fold was tentatively identified [Figure 17; (260)]. Furthermore, from an alignment of these two enzymes with 1 8 nucleotide-binding proteins with known structures, four additional f3-strands were predicted, thus generating a putative six-stranded nucleotide-binding sheet (260). There seems little doubt about the initial f3af3 fold, but the assignment of the four other f3-strands should be considered speculative. Clearly, the determination of the three-dimensional structure of one (or both) of these enzymes would be of considerable interest. NOS and OCS are unusual in the sense that both enzymes use either NADPH or NADH as cofactor. In this context, OCS has the fingerprint of NADP(H)-linked dehydrogenases (GXGXXA) , but NOS has the GXGXXG motif usually seen in NAD(H)-linked dehydrogenases (Figure 17). NOPALINE AND OCTOPINE SYNTHASES

Xuan et al (201) recently reported a weak resemblance between the fingerprint of Wierenga et al (264) and the Y.

SACCHAROPINE DEHYDROGENASE


549


lipolytica SDH segment between residues 195 and 230. Indeed, inspection of the sequence does not reveal the core fingerprint for the binding of NADH. The sequence GSG that is centered around Ser207 may be involved in the binding of the dinucleotide pyrophosphate group, but the characteristics of a typical f:!af:!-binding fold are not readily discemable in this segment (Figure 17). However, it may be noted that if the sequence Gly203-Gly208 is reversed, the sequence GXGXXG is apparent.

REACTION MECHANISM(S) AND CATALYTIC DOMAINS Attempts have been made to associate specific amino acids , or particular regions, with catalytic function of the N-(CA)amino acid dehydrogenases. Since the dinucleotide-binding domains of the opine dehydrogenases, NOS and OCS, are located near the NHrtermini, it has been suggested (260) that residues involved in catalysis are located in the C-terminal moieties of these crown gall enzymes. The reaction mechanism of NOS and OCS [and the other N-(CA)amino acid dehydrogenases] involves the binding of an a-keto acid, and it is tempting to draw analogies with the reactions mediated by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH). The active sites of the latter enzymes contain Asp, His, Arg (and other) residues (283, 284). Of these residues, the Asp-His pair has been shown to be part of a proton relay system (285, 286), and Arg stabilizes the a-keto acid by providing a two point interaction with the carboxylate group of this acid (287). In LDH and MDH , the catalytically relevant Asp and Arg residues are contained in a conserved Asp-Xaa-Xaa-Arg sequence. Monneuse & Rouze (260) noted that such a sequence is also present in conserved segments in the C-terminal moieties of both NOS (residues 284-287) and OCS (residues 235-238). These authors also suggested that His373 of NOS, and His263 of OCS, could be part of a putative protein relay system in the opine dehydrogenases. However, these residues are not homologous in the alignment proposed by these authors, whereas His373 of NOS and His333 of OCS are contained in homologous sequences. Furthermore, His288 of NOS and His338 of OCS should also be considered as relay candidates, particularly since they are contained in a highly conserved sequence of 11 residues. In both CEOS and SDH, the nucleotide-binding region is centrally located, and the dot-plots (Figure 16) indicate homology between the N- and C terminal regions of these proteins. The sequence, Asp-Xaa-Xaa-Arg, is also found in the C-terminus of CEOS (residues 300-303), and two such sequenc es (at positions 127-130 and 211-214) are present in SDH. Chemical modification and inhibitor studies indicate participation of Lys, His, and Arg residues in SDH catalysis (Refs. 288, 289, 290, respectively). From these data Fujioka (291) has proposed an attractive mechanism for


550


saccharopine biosynthesis , which may also be applicable to the synthesis of W-(CE)ornithine by CEOS . According to this model, the first step in the synthesis of saccharopine involves hydrogen bonding between a lysyl residue in the enzyme and the carbonyl oxygen of a-ketoglutarate . Protonation of the oxygen atom, and nucleophilic attack by the E-NH2 group of lysine (substrate) on the a-keto carbon, yields a carbinolamine. Migration of a proton from the secondary amine to the -OH group of the carbinolamine causes elimination of water, and the formation of a Schiff base intermediate . The positively charged imine nitrogen facilitates the stereospecific (A-side) transfer of hydride ion, from NADH to C-2 of the I ,3-dicarboxypropyl moiety of the Schiff base, to yield saccharopine. Now that the SDH gene has been cloned, it should be possible to test this model by site-directed mutagenesis.

CONCLUSIONS AND FUTURE DIRECTIONS Octopine was isolated by Morizawa in 1 927 , and the first crown gall opines were described in the late 1 950s and early 1 960s. At the time of their discovery, these unusual N-(CA)amino acids were primarily of academic interest and were considered "curiosity" chemicals. However, the continued study of these compounds has had ramifications for many areas of biochemis try, physiology, agriculture, and medicine. The "trapping" of N-(CA)amino acids has provided insight into the mech anisms of the reactions catalyzed by aldo1ases, amino acid oxidases, and pyruvoyl-linked amino acid decarboxylases (2 1 4-2 1 8) . The discovery of these amino acids in marine invertebrates provided new (and unexpected) perspectives concerning the regulation of glycolysis and the anaerobic physiology of these species ( 1 5G-1 53). The A . tumefaciens opines have been used as "reporter" molecules, and the oes and nos promoters have been incorporated into T-DNA vectors for the improvement of crops and plants by genetic engineering (292, 292a, 293). Last but not least, N-(CA)amino acids have been instrumental in the design of drugs for control of hypertension in humans (229-23 1 ) . The discovery i n the mid- 1 980s o f the N W-(CA)amino acids i n bacteria (L . laetis) was accidental , and their functions are presently unknown. One won ders if these compounds are involved in the regulation of gene expression, or whether they are allosteric modifiers of enzyme activity. Are they in termediates of as-yet-unidentified biosynthetic pathways, or precursors to more complex molecules, e . g . bacteriocins? Are they, perhaps , simply de toxification products? From our experience, it seems probable that N (CA)amino acids have been overlooked in other bacterial species, and careful re-examination may reveal these compounds to be more widespread than currently envisaged. Such studies may also yield clues to the function(s) of



55 1

these metabolites in L. iactis, and previous experience suggests surprise may be in store ! The N-( CA )amino acid synthases (EC 1 . 5 . 1 -) act on the CH -NH group, and the amino acid dehydrogenases (EC 1 .4. 1 -) on the CH-NH2 group of donors. In the reverse direction, these enzymes use NAD(P)H, a-keto acids, and amino acids (or NH3) as substrates. One is tempted to speculate on the structural and catalytic similarities of these enzymes, and ponder whether the respective genes were derived from a common progenitor. The future de termination of the structures of these proteins by X-ray diffraction, or NMR spectroscopy , will be of considerable interest and should provide details of the nucleotide-binding and catalytic domains of these (three-substrate) enzymes . It augurs well for investigators that there is much to learn , and many questions to be answered, in this area of amino acid biochemistry. ACKNOWLEDGMENTS

Professors Emeriti Frank C. Happold of Leeds University, England, and Robert A. MacLeod of McGill University, Montreal, taught J . T. the "ways" of science. Chuck Wittenberger, Jack Folk , and Alton Meister provided the encouragement to take the "road less-travelled." We acknowledge the ex cellent technical assistance of Robert Harr and the expert help of Charlette Cureton in the preparation of this review. Finally, we thank Stephen Miller, Stanley Robrish , John Wootton, and our many colleagues at the National Institutes of Health, for their contributions, advice, and criticisms .

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