Gene, 91 (1990) 27-34 Elsevier

27

GENE 03582

The methane monooxygenase gene cluster of Metlirylococcus capsulatus (Bath) (Recombinant DNA; methanotroph; iron-sulfur flavoprotein; nucleotide sequencing; hemerythrin; ribonucleotide reductase)

A.C. Stainthorpe, Veronica Lees, George P.C. Salmond, Howard Dalton and J. Colin Murrell Departmentof BiologicalSciences, Universityof Warwick,Coventry,CV4 7AL (U.K.) Received by S.G. Oliver: 8 August 1989 Revised: 7 December 1989 Accepted: 11 December 1989

--

-

SUMMARY

Methane is oxidised to methanol in methanotrophic bacteria by the enzyme methane monooxygenase (MMO). Methylococcus capsulatus (Bath) produces a soluble MM0 which oxidises a range of aliphatic and aromatic compounds with

potential for commercial exploitation. This multicomponent enzyme has been extensivelycharacterised and biochemical data have been used to identify a 12-kbfragment of Methylococcus DNA carrying the structural genes mmoY and mmo2, coding for the fl- and y-subunits of MM0 component A, the methane-binding protein. We now report the complete nucleotide (nt) sequence of mmoX, the gene encoding the a-subunit of component A which is found to be 5’ to mmoY and mmoZ. We also report the complete nt sequence of mmoC which encodes component C, the iron-sulfur flavoprotein of MMO, the N terminus of which is significantly homologous with spinach ferredoxin. The mmo structural genes are clustered within a 7-kb region and are closely linked to two small open reading frames of unknown function.

INTRODUCTION

Methylococcus cupsulatus (Bath) is an obligate methanotroph deriving its carbon and energy solely from the oxidation of methane to carbon dioxide. Methane is oxygenated to methanol by MMO. This enzyme catalyses the insertion of oxygen into methane and a wide variety of aliphatic, alicyclic and aromatic compounds (Colby and Dalton, 1978; Stirlmg and Dalton, 1979). CorrespondenceIO:Dr. J.C. Murrell, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL (U.K.) Tel. 0203-523553; Fax 0203-523701. Abbreviations: aa, amino acid(s); bp, base pair(s); d, deletion; DEAE, diethyl amino ethyl; EPR, electron paramagnetic resonance; EXAFS, extended x-ray absorption fine structure; kb, kilobase pairs or 1000 bp; M., Melirylococcus;MMO, methane monooxygenase; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF or orf (for gene), open reading frame; PAGE, polyacrylamide-gel electrophoresis; RF, replicative form; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI/O.O15mM Na, 0citrate pH 7.6; [ 1,denotes plasmid-carrier state. 0378-l 119/90/%03.50

0

1990 Elsevier

Science Publishers

B.V. (Biomedical

Division)

The soluble MM0 complex of M. cupsulatus has been resolved into three Components (A, B and C) by DEAEcellulose chromatography (Colby and Dalton, 1978).The 210-kDa protein A, the site of hydrocarbon oxygenation, comprises tandem copies of the three subunits a, /I and ‘y, of 54,44 and 20 kDa, respectively (Woodland and Dalton, 1984). Two iron atoms per molecule of protein A form a Cc-hydroxobridge structure believed to be the site of oxygenase activity (Ericson et al., 1988).Components B and C are single-subunit proteins of 16 and 38 kDa, respectively. Protein B regulates oxygenase activity (Green and Dalton, 1985). Reducing equivalents are passed from NADH to component A by the iron-sulfur flavoprotein component C (Lund et al., 1985).All three components are necessary for the conversion of methane to methanol. Understanding of the regulation and structure-function relationship of methane metabolism enzymes would be greatly enhanced by analysis of the genes encoding and controlling them. Classical genetic studies of methanotrophs have been hindered in the past by the absence of a

28 (b) I d e n t i f i c a t i o n o f mmoC

system of genetic exchange (Williams and Bainbridge, 1971) and the difficulty in obtaining mutants (Williams et al., 1977). Recent methods for the transfer of broad-hostrange plasmids into methanotrophs (AI-Taho and Warner, 1987) have had only limited success when applied to M. capsulatus (J.C.M., unpublished). Analysis of the methane metabolism genes by molecular cloning techniques has proved a more productive approach. Oligo probes specific to the N-terminal aa sequence data available for the soluble MMO polypeptides have been used to identify recombinant plasmids encoding mmo genes. The gene encoding the ?-subunit (mmoZ) was cloned and the fwst 57 nt of the sequence were reported (Mullens and Dalton, 1987). In a previous paper we described the cloning of the mmo Y gene (coding for the/~-subunit of protein A), which was found to be closely linked to mmoZ. We also reported the complete nt sequence of both genes and a restriction map of the recombinant plasmid pCH4 containing these genes (Stainthorpe et al., 1989). The aim ofthe present study was the identification and complete nt sequence of the mmoX gene (which encodes the 0~-subunit) and mmoC (encoding component C), which, together with mmoY and mmoZ, form a closely linked gene cluster.

Plasmid pCH4 consists of an 11.9-kb EcoRl fragment of M. capsulatm (Bath) DNA cloned into the EcoRl site of pBR325 (see Fig. 2A; Stainthorpe et al., 1989). Restriction digests of this plasmid, prepared by the alkaline lysis method were resolved on a 1.0% agarose gel and transferred by Southern blotting to nitrocellulose (Maniatis et al., 1982). The mmoC-specific oligos (see Fig. lb) were end-labelled by the addition of 32p using [~,-32p]ATP ( > 185TBq/mmol) and "1"4 polynucleotide kinase. The restricted DNA bound to nitrocellulose was challenged with labelled probe ( > 2 × l0 T cpm/ml) in Denhardt's solution containing 100/Jg denatured herring testis DNA per ml and 6 × SSC for 18 h at 20°C (Maniatis et al., 1982). The melting temperature for the mmoC oligo probe was estimated to be 68°C (Lathe, 1985). Filters were washed for 15 rain in 6 × SSC at 20°C and then subjected to successive 15-min washes in 6 × SSC at 10°C increments up to 90 ° C. Autoradiography was carried out after each temperature increase to reveal any bound DNA-probe heteroduplex formations.

RESULTS AND DISCUSSION MATERIALS AND METHODS

(a) N-terminal aa sequence o f c o m p o n e n t C and c o n s t r u c tion o f C-specific oligo p r o b e The N-terminal aa sequence of purified component C and ~-subunit of protein A was determined as described previously (Stalnthorpe et al., 1989). The first 16 N-terminal aa of protein C were identified (Fig. la). The first six N,terminal aa of the a-subunit were found to be Met-LysLeu-Ser-Thr-Asp. The average ~o G + C of M. capsulatus DNA has been determined, directly for the mmoY and mmoZ genes (Stainthorpe et al., 1989) and indirectly from DNA melting experiments, to be 61% and 62.5%, respectively. This ~o

(a) Materials

Restriction endonucleases, DNA ligase and radiolabelled deoxyribonucleotides were supplied by Amersham International and used according to the suppliers' recommendations. DNA polymerase was obtained from Gibco-BRL. All other chemicals and reagents were supplied by Sigma and were of the highest purity available. Oligos for use as probes and sequencing primers were synthesised on an Applied Biosystems DNA synthesiser and purified by highperformance liquid chromatography.

10

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ATG CAG CGA GTT CAC ACT &TC ACG GOG GTG &OG GAG ~lkT GGO GP.~ TCG

Fig. I. ProteinC structure.(a) The aa sequenceofthe N-terminalregionofsoluble MMO componentC, as determinedby automatedgas phase Edman degradation after purificationby the procedureof Colby and Dalton(1978).(b) The nt tripletsfor the N-terminalaa sequence.The boldfacent are those used in the reverse translation to a complementaryoligo probe. Note the use of G or C in certain degenerate positions; see RESULTS AND DISCUSSION,sectiona, (¢) The nt sequenceofthe 5' end of the mmoCgene.The sequencedepictedin boldface matchesthe probe sequenceperfectly, despite the selectionof G or C in degeneratepositions. The underlinednt indicate the position of ~.possible inversionwithin the second codon; see RESULTS AND DISCUSSION,section e.

29

(c) Sequencing of mmoC and mmoX

G + C richness places restrictions on ORF codon usage with G or C selected predominantly in degenerate positions (Stainthorpe et al., 1989). A region of the known protein C N-terminal aa sequence was chosen (aa 9-15) (Fig. lb), which reverse translated to nt sequence of relatively low degeneracy. Arbitrary selection of G or C in certain degenerate positions made it possible to design a mixed oligo probe of 128 instead of 2048 different sequences for the seven codons (bold type in Fig. lb).

The location and primary sequence of mmoC was determined by sequencing the 1612 bp region of cloned Methy!ococcus DNA 3' to the mmoZ gene bounded by the restriction sites Pstl and Sail (Fig. 2a). The DNA was sequenced, in both orientations, by the strategy adopted in Fig. 2b. The contiguous nt sequence of 1612 bp obtained encodes two ORFs. The first ORF of 312 nt (104 aa, predicted Mr 11943), has no known function but separates mmoZ from mmoC. The second ORF, mmoC, of 1047 nt translates to a polypeptide of 349 aa. Of the first 16 aa of this polypeptide 15 match the N-terminal sequence obtained from the purified component C. The mrnoC gene codes for a polypeptide of 38 581 Da which is in very close agreement with the component C polypeptide of 39kDa estimated by SDS-PAGE (Colby and Dalton, 1978). The nt sequence encodes Glu at position 2, whereas aa analysis identified a Thr residue at this position. Since this region of DNA has been sequenced on both strands from independent isolates of the mmoC gene from the same laboratory strain of M. capsulatus (Bath), we can only assume that either the aa sequence analysis at position 2 was incorrect or that a mutation has since arisen at the second codon of mmoC.

(b) Location of mmoC on plasmid pCH4 A restriction map of the l l.9-kb EcoRl fragment of Methylococcus chromosomal DNA contained on plasmid pCH4 (Fig. 2) was reported previously (Stainthorpe et al., 1989). Restriction digests of pCH4 were transferred to nitrocellulose and probed with radiolabelled oligos specific for mmoC to locate the 5' end of the mmoC gene. After hybridisation, stringent washing (70°C in 6 x SSC)localised the 5' end of mmoC gene to the 0.7-kb Pstl fragment (Fig. 2a). "i'he 3' end of the mmoY gene extends 18 bp into this restriction fragment thus confirming that mmoC is closely linked to the mmoY and mmoZ genes on the cloned region of the M. capsulatus chromosome (Figs. 2 and 3).

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Fig.2. +Restriction maps of the mmo region. (a) Restriction map of the I 1.9-kb insert of M. capsulatus (Bath) DNA of plasmid pCH4. Based on the aa sequence data of the MMO polypeptides we have identified by oligo probing and nt sec~uencing(see RESULTS AND DISCUSSION, section e and Stainthorpe et al., 1989), the exact location of the mmo genes encoding those polypeptides. Transcription of the mmo genes proceeds 5' to 3', left to right in the figure.(b) Enlarged restriction map of the 1612-bp Pstl-$all fragment encoding mmo¢~ and orJY.Arrows indicate the direction and extent of sequence determination of individual clones. DNA fragments generated at the strategic restriction sites or random fragments generated by partial digests with Sau3A or Taql were cloned into the RF DNA of MI3tgl30 and MI3tgl31 (Kieny et al., 1983). At~er subsequent transfection into E. coli strain TGI (supE, thi, A(lac.pro), hsdDS[F' traD36, proA +B ÷, lacl q lacZAMl5], MI3 recombinants were sequenced by the dideoxy method (Sanger et al., 1977) using [a-35S]ATP (> 3"/TBq/mmol). Sequence data of the regions in b and e were compiled by sequencing both strands at least twice and up to six times in some regions. Analysis of nt sequences and derived aa sequences was performed using the Microgenie sequence analysis programme distributed by Beckman Instruments Inc. and protein analysis via the Dap search programmes (Collins and Coulson, 1987). (e) Enlarged restriction map encompassing the 3142-bp region encoding the mmoX gene. Arrows indicate the direction and extent of sequence determination. The location of the sequence homologous to the unique sequence primers indicated by an open circle and the arrows indicate the sequence derived from them. The sequence derived from the random clones is represented by a solid line with terminal inward pointing arrows. Sequence across restriction sites and gaps in schemes b and e was derived from random fragments generated by partial digests with Sau3A and Taql.

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Fig. 3. The nt sequenceof the 1612-bpPstl-$all fragment of plasmid pCH4 insert DNA. The fragmentcarries ~ene mmoCencodingthe soluble MMO component C. An ORF of unknown function,offY, lies between mmoZ, the 3' 18 bp of which are shown, and mmoC. The coding sequence is depicted above the derivedaa sequence.PutativeShine-Dalgarnosequenceand start codons are depictedin bold type.The mmoCgenestarts at nt 498 and extends to ! 544, orfl' from 173-485. Asterisksindicate the stop codons. Last digit~ of numbers are aligned with corresponding nt. Corresponding aa is below the second nt of each codon.

The primary sequence of mmoX, the gene encoding the 0t-subunit of component A, was determined by sequencing the EcoRI-Clal region of Methylococcus DNA 5' to the mmoY gene. The sequencing strategy adopted was as shown in Fig. 2c. The contiguous nt sequence of 3142 bp encodes an ORF of 1581 nt which translates to a single polypeptide of 527 aa with a predicted Mr of 60 636. This is in close agreement with the published size of the purified u-polypeptide obtained by S D S - P A G E (Woodland and Dalton, 1984). Four ofthe first 6 aa of this ORF match the known N-terminal aa sequence data from the purified polypeptide. The two aa sequence discrepancies could result from the difficulties in differentiating these residues by the chemical sequencing method used. Although the known sequence was relatively short (6 aa), the likelihood of this arrangement of 4 aa occurring randomly in an N-terminal 6-an sequence, combined with the ORF size and its proximity to the other mmo genes leads us to believe the 0c-subunit is encoded by this gene. Sequence analysis Cell extracts of Methylococcus always appear to contain stoichiometric amounts of protein A subunit polypeptides

(d)

at high concentrations (Woodland and Dalton, 1984) suggesting that co-transcription occurs. This report confirms that genes encoding the ~-, r- and ?-subunits of protein A, mmoX, mmoY and n,,moZ are contiguous on the Methylo. coccus chromosome (Fig. 2). Intercistron regions are small, the largest, between mmoX and mmoY being 78 bp long (Fig. 2). The mmoY and mmoZ genes are separated by an ORF, orfX, of unknown function (Stalnthorpe et al., 1989). A second ORF, off Y, separates the structural genes for the protein A subunits from the protein C structural gene mmoC (Figs. 2 and 3). Sequences capable of forming stem-andloop structures in mRNA have been found between mmoX and mmoY and between mmoZ and orfY (Stainthorpe et al., 1989). Ofthe genes in the MMO cluster, only mmoX is preceded by 5' sequence homologous to Escherichia coli -35 and -10 consensus sequences (Fig. 4). Sequence homologous to the -12 and -24 conserved regions of Pseudomonas (Deretic et al., 1987) and to n/f- and ntr-like consensus promoter regions (Dixon, 1986) are also found in the A + T-rich sequence 5' to the mmoX gene (Fig. 4). The promoter sequences which operate transcription of these highly expressed copper- and methane-regulated proteins in Methylococcus may be entirely novel. Further inves-

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~c~C~t`CTGTC~Nk°TGAT~C~A`%CTG~A~CTG~°~&~TGW1~G~CJ~A~Ac~CTG&TC°~C&°C~A~TcC°~AC~~AC °

a

E

L

S

e

V

l

A

B

L

H

O

L

g

S

U

G

K

W

L

l

A

Q

P

H

V

a

G

D

K

L

W

T

3050 3109 GT1~GG&~&TATCAN~cTGAACTG~GTCTTcAAGAA~C~GGTGM`G~C&TTCAATTGAM`C°°G~~C~°TC&CAG°GCG~°~CCC°&C°C L D D l K it L N C V F K N P V K A F N ACO&TCGTTCG&TCM,CCTCAkACCAA,tAAGGAACATC01t~ N mmo¥...

Fig. 4. The nt sequence of the 3143 bp region ofplasmid pCH4 insert DIqA. The fragment carries gene mmoX encoding the soluble MMO component A ~-subunit.The coding nt sequence is depicted above the derived aa sequence and the putative Shine-Dalgarno sequence is boxed. The Cial restriction site at 3137 bp contains the first 2 nt of the start codon of mmo ¥, the p-subunit gene (Stainthorpe et al., 1989). The ,nderlined sequences show significant homology to the promoter sequences from Pseudomonas (Ps -24 and Ps -12) (Deretic et el., 1987),E. colt tEc -35 and Ec -10), and to the nil and ntr.like promoter regions (niD (Dixon, 1986). The mmoX gene starts at nt 1479 and extends to nt 3062, Asterisks indicate stop codons. Last digits of numbers are aligned with corresponding nt. Corresponding aa is below th~ second nt of each codon. tigation by S 1 nuclease mapping or primer extension experiments are needed to resolve the structure of transcription control elements of M M O . The codon usage is strongly biased towards the use of G or C in the degenerat~ positions of the O R F s (Table I). Average ~ G + C o f m m o X at 6 0 ~ is slightly lower than both the 6 1 ~ previously reported for m m o Y and m m o Z

(Stainthorpe et al., 1989). Each coding sequence is preceded by an in-frame A T G start codon and a putative Shine-Dalgarno sequence (Figs. 3 and 4). (e) Analysis of deduced polypeptides Protein homology searches of the m m o X and orfY products using the Beckman Microgenie database and the

32 TABLE 1 The nucleotide usage in mmoX, mmoC and orfl' codons

G + C contenta Average of mmoC

oq'Y

57 40 83 60

62 44 82 63

64 43 69 59

60 40 83 60

60 41 83 61

527

348

103

558

1433

1st position 2rid position 3rcl position Average Number of codons

All mmo genes

mmoX

mmo Y + mmoZ

Calculatedusingthe BeckmanMicrogenieprogramme.The data on mmoY and mmoZ are derivedfrom Stainthorpeet al. (1989). Data on mmoX, mmoC and orfF are from Figs.3 and 4. DAP search programme (Collins and Coulson, 1987)failed to detect any significant homology to any proteins in these databases. The protein predicted by m m o C , however, showed significant homology at the N terminus (44% over 90 aa) with ferredoxins of plant, cyanobacterial and archaebacterial origins (Takahashi et al., 1983; Hase et al., 1978; 1982). Two strongly conserved domains were common to the ferredoxins and protein C (Fig. 5). Three of the four Cys residues of the 2Fe-2S iron-sulfur centre of ferredoxins occur within the first conserved domain (aa 40-51; Fig. 5). The fourth Cys (protein C, aa 81) lies in the second conserved domain (aa 74-86; Fig. 5), of protein C. We have therefore confirmed that protein C has a plant-like ferredoxin 2Fe-2S centre as suggested by its absorbance and EPR spectra (Lund et ai., 1985). Protein C also shows significant homology to NADH. cytochrome bs reductase from human erythrocytes (Yubisui

eta]., 1984; Fig. 6a). Both enzymes contain l mol FAD/mol protein which accepts reducing equivalents from NADH. There must therefore be domains within both proteins which interact with N A D H and FAD. The two highly conserved domains are apparent (aa 221-235 and 310-324 protein C, Fig. 6a), which may be ADP-binding domains common to protein C and NADH-cytochrome bs reductase. The ADP-binding domains of many flavoproteins exhibit a characteristic/~-~-/~ fold (Wierenga et al., 1986) centred around a G X G X X G 'core fingerprint' (where X is any aa). Sequences resembling this *core' arrangement do not appear in either protein C or cytochrome bs reductase. An alternative ADP-binding domain may centre around the G G T G domain common to both proteins at residues 223 of protein C (Fig. 6a). These possibilities await further detailed biochemical analysis for verification. The role of protein C as a short electron transport chain

10

protein c

ORvnT

20

x

30

40

xan sonnv

European elder

A S ~ L ~ T P i ~ Q E F ~ C P D DV~ZLEHAEELGZDIP¥~C] White pop,nat &F~LLTPD6~KEFIh?~ D DV~XLDOAEELGIELP~a[I~ Horsetails &¥KTVLKTPS6BFTLDVPEGTTZLDN~F~GYDLPFa~J Nostoc muscorum &~LVI~GTETTIDVPDDB~ I L D ~ L P ~ _ f ~ I = J Spinach AT ¥1¢_V~LV~PS~SQV I~q~DDB¥ ZLDAkgEK(~DLP¥~ 50 protein C European elder White poptnac Horsetails

60

~?~~KLV'AGSV~SnQ8 na~SKLVZaDL~S~S

?0

80

90

rr~Dsoxss~:i~zan~xn

FLDDZ~XZZaWL~aAreaSO_VVm

~~KVVSGSVnQSEGar ~nG2Hmm~v~az~ssn,.v~n x~onox~o~Wl~Tsnr~xn aaq~ga~grZsGsv~s~sr ~o~t~mw~Iz~T.~_v,rxs

Nostoc mus¢orum _ ~ . q R x v s 6 ~ , n o s ~ u

Spinach

Fig. 5. Alignmentof the N-terminal92 aa of protein C with the 2Fe2S ferredoxinof European elder (Takruri and Boulter, 1979),white popinac (Benson and Yasanobu, 1969),horsetails(Hase et ai., 1977),Nostoc muscorum (Haseet ai., 1982)and spinach(Takahashiet al., 1983).Stronglyconserveddomains (around the Cys residues of the iron-sulfur centre) are boxed, identities are depicted in bold face and conserved substitutions are underlined. The numberingcorresponds to protein C with l-aa gaps after positions 26 and 28 to allow alignmentwith other sequences. Gaps in other aa sequences are introduced to allow alignment with protein C. The homology of protein was initiallyidentified using the Beckman 'Microgenie'programme.

33 (a)

221 153

Z~LAPWSMV~MQEWTAPNETRIYFGVN~PELF~IDELKSLERSMRNLTVKA *11111"*1"* **1 I * ** I* II ** II I I**** I VRGGTGI~MLQVIRAIMKDPDDHTVCHLLFANQTEKDILLRPBLEELRNF~SARFKL

~v'~HPSGD WE ~ a S P I D A L R E D L E S S D A N P D I Y L ~ P P ~ I D A ~ E L V R S R *

*

I

I

It

*

*

I*

I ***

*1

* *1111

338

GIPGE~W

I1" II

I

I

I

W~TLDRAPEANDYG~FVNEEMIRDHLPPPEEEPLVLM~PPPMZQY~LPHLD~TBR~

I

2?3

(b)

Protein

aa

aa Sequence

a subunit

135

NGYLAOVLDEIR~THQ

a

subunit

235

VFLSIETDELR~IANG ww

ribonuoleotide reductase

230

w

KI IRLIAR~AL~LTGTOHML

Fig. 6. The aa sequence homologies. (a) The aa sequence homology comparison of protein C (top lines) with NADH-cytochrome bs reductase of human erythrocytes (bottom lines)(Yubisui et al., 1984). Identities are in boldface and connected by dashed lines, conserved domains are underlined and asterisks between the sequences indicate conserved substitutio.s. This portion of the aa sequence of protein C is derived from nt 1158-1511 (Fig, 3). (b)The aa sequence homology between the a-subunit of protein A of MMO and the highly conserved region of the ribonucleotide reductase B 2 protein of E. coll, Asterisks denote identities between potential iron ligands of ribonucleotide reductase and the a-subunit. Numbers indicate the position of aa sequence of protein A a-subunit and ribonu¢leotid~, reductase compared here. Regions forming a-helix are underlined. This portion of the aa sequence of protein A a-subunit is derived from nt 1880-1927 and 2180-2227 (Fig. 4).

is a feature common to many other multicomponent aikane and aromatic oxygenases. Two redox centres are commonly used in the transfer of electrons from the electron donor to the oxygenase component. The two redox centres may be contained on two separate components, as for the NADH-ferredoxin reductase of benzene dioxygenase of Pseudomonas putida (Irie et al., 1987), or together on one component as in protein C and benzoate dioxygenase NADH-ferredoxin reductase (Yamaguchi and Fujisawa, 1978). On the basis of size and structural organisation, protein C most resembles the NADH-ferredoxin reductase of the benzoate dioxygenase system. Once this component is sequenced it will be possible to make a more detailed comparison. The mmo gene encodes the 0c-subunit of soluble MMO protein A. Studies with radiolabelled acetylene, a suicide substrate for the enzyme, have shown the active site to reside on this subunit (Prior and Dalton, 1985), which suggests that the/~-hydroxo-bridge cluster of the active site is closely associated with this polypeptide (Green and Dalton, 1988; Ericson et al., 1988). This type of iron centre occurs in proteins of diverse function including hemerythrins (Kurtz etal., 1977); ribonucleotide reductases (SjOberg et al., 1987) and purple acid phosphatase (Davis and AveriU, 1982). EXAFS data on protein A show that the

iron centre is similar to that of hemerythrin with a shell of three Fe-O and two Fe-N bonds around each iron (Ericson et al., 1988). By analogy with hemerythrin, the iron centre ligands of protein A could be four His residues and two bridging protein carboxylates. As protein A is composed of tandem copies of three subunits, the iron centre may lie at an interface between two or more ofthe subunits and exhibit twofold symmetry with respect to the ligands. The origin of the iron centre ligands has yet to be determined but the close association to the 0c-subunit suggests it may donate one or more iron-binding ligands. The conserved residues Glu ~s, His ~s, Asp237, Glu23s and HisTM of each B2 subunit of ribonucleotide reductase have been proposed as potential ligands of the iron centre (Carlson et al., 1984; Sj0berg et al., 1985; 1987). The B2 protein and the 0~-subunit of MMO show no general homology but there are two regions of the latter which are similar to the conserved sequence of the former (Fig. 6b; Sj0berg et al., 1985). The predicted tertiary structure for these regions suggest that both these histidine residues of the ~-subunit are located in 0~-helixes and that an aspartate or glutamate group is located one turn earlier in the helix (Fig. 6b). This situation is matched in ribonucleotide reductase (Fig. 6b; SjOberg etal., 1985; Carlson et al., 1984). The spatial arrangement is such that it is possible for both the His and the Asp or Olu residues

34 to ligate to the iron centre (Fig. 6b). A similar observation has been made in hemerythrin (Stenkamp et al., 1984; Kurtz et al., 1977). There are, however, no further regions of homology between the 0c-subunit and the B 2 protein or between the 0c-subunit and hemerythrin. By the use of sitedirected mutagenesis, it should be possible to determine whether the residues discussed figate the iron and whether they are obligatory for enzyme activity of MMO.

ACKNOWLEDGEMENTS

We thank Gill Scott for the preparation of the C-specific oligos and sequence primers, and the Gas Research Institute (Chicago) for their financial support through Contract No. 5086-260-1209. The authors also acknowledge Gerald Chapman for purification of the ~-subunit of MMO. REFERENCES AI-Taho, N.M. and Warner, P.J.: Restoration ofphenotype in Escherichia coli auxotrophs by pULBll3-mediated mobilization from methylotrophic bacteria. FEMS Mierobiol. Lett. 43 (1987) 235-239. Benson, A.M. and Yasanobu, K.T.: Non-beam iron proteins XI: some genetic aspects. Proc. Natl. Acad. Sci. USA 63 (1969) 1269-1273. Carlson, J., Fuchs, J.A. and Messing, J.: Primary structure of the Escherichla coil ribonucleoside diphosphate reductase operon. Proc. Natl. Acad. Sci. USA 81 (1984)4294-4297. Colby, J. and Dalton, H.: Resolution of the methane monooxygenase of Methylococcus capsulatus (Bath) into three components: purification and properties of component C, a flavoprotein. Biochem. J. 171 (1978) 461-468. Collins, J.F. and Coulson, A.F.W.: Molecular sequence comparison and alignment, in Bischop, M.J. and Rawkins, C.J. (Eds.), Nucleic Acid Sequence Analysis: A Practical Approach. IRL Press, Oxford, 1987, pp.323-358. Davis, J.C. and Averili, B.A.: Evidence for a spin-coupled binuclear iron unit at the active site ofthe purple acid phosphatase from beefspleen. Proc. Natl. Acad. Sci. USA 79 (1982) 4623-4627. Dereti¢, V., Gill, J.F. and Chakrabarty, A.M.: Alginate biosynthesis: a model system for gene regulation and function in Pseudomonas. Biotechnology 5 (1987)469-477. Dixon, R.: The xyMBC promoter from the Pseudomonas putida TOL plasmid is activated by nitrogen regulatory genes in Escherichla coll. Mol. Gun. Genet. 203 (1986) 129-136. Ericson, A., Hedman, B., Hodgson, K.O., Green, J., Dalton, H., Bentson, J.G., Beer, R.H. and Lippard, SJ.: Structural characterization by EXAFS spectroscopy of the binuclear iron centre in protein A of methane monooxyg~nase from Methyiococcus capsulatus (Bath). J. Am. Chem. Soc. !!0 (1988) 2330-2332. Green, J. and Dalton, H.: Protein B of soluble methane monooxygenase from Methyiococcus capsulatus (Bath): a novel regulatory protein of enzyme activity. J. Biol. Chem. 260 (1985) 15795-15801. Green, J. and Dalton, H.: The biosynthesis and assembly of protein A of soluble methane monooxygenase of Methyiococcus capsulatus (Bath). J. Biol. Chem. 263 33 (1988) 17561-17565. Hase, T., Wada, K. and Matsubara, H.: Horsetail Equisetum telmateia ferredoxins I and II. J. Biochem. 82 (1977) 267-276. Hase, T., Wakabayashi, S., Matsubara, H., Kerscher, L., Oesterhelt, D., Rao, K.K. and Hail, D.O.: Complete amino acid sequence of Halobacterium halobium ferredoxin containing an N '~ acetyllysine residue. J. Biochem. 83 (1978) 1657-1670.

Hase, T., Matsubara, H., Hutber, G.N. and Rogers, LJ.: Amino acid sequences of Nostoc strain MAC ferredoxins I and II. J. Biochem. 92 (1982) 1347-1355. Irie, S., Doi, ~. Yorifuji, T., Takagi, M. and Yaro, K.: Nucleotide sequencing ~id characterization of the genes encoding benzene oxidation enzy,.~es of Pseudomonas putida. J. Bacteriol. 169 (1987) 5174-5179. Kieny, M.P., Lathe, R. and Lecocq, J.-P.: New versatile cloning and sequencing vectors based on bacteriophage MI3. Gene 26 (1983) 91-99. Kurtz, D.M., Shriver, D.F. and Klotz, I.M.: Structural chemistry of hemerythin. Coordin. Chem. Rev. 24 (1977) 145-178. Lathe, R.: Synthetic oligonucleotide probes deduced from amino acid sequence data; theoretical and practical considerations. J. Mol. Biol. 183 (1985) 1-12. Lund, J., Woodland, M.P. and Dalton, H.: Electron transfer reactions in the soluble methane monooxygenase of Methylococcus capsulatus (Bath). Eur. J. Biochem. 147 (1985) 297-305. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, NY, 1982. Mullens, I.A. and Dalton, H.: Cloning of the gamma-subunit methane monooxygenase from Methylococcus capsulatus. Biotechnology 5 (1987) 490-493. Prior, S.D. and Dalton, H.: Acetylene as suicide substrate and active site probe for methane monooxygenase from Methylococcus capsulatus (Bath). FEMS Microbiol. Lett. 29 (1985) 105-109. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. SjOberg, B.-M., Sanders-Loehr, J. and Loehr, T.M.: Identification of a hydroxide ligand at the iron centre of ribonucleotide reductase by resonance Raman spectroscopy. Biochemistry 26 (1987)4242-4247. Stainthorpe, A.C., Murrell, J.C., Saimond, G.P.C., Dalton, H. and Lees, V.: Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath). Arch. Microbiol. 152 (1989) 154-159. Stenkamp, R.E., Sieker, L.C. and Jensen, L.H.: Binuclear iron complexes in methemerythrin and azidomethe merythrin at 2.0-A resolution. J. Am. Chem. Soc. 106 (1984) 618-622. Stirling, D.I. and Dalton, H.: The fortuitous oxidation and cometabolism of various carbon compounds by whole-cell suspensions of Methylococcus capsulatus (Bath). FEMS Microbiol. Lett. $ (1979) 315-318. Takahashi, Y., Hase, T., Wada, K. and Matsubara, H.: Ferredoxins in developing spinach cotyledons; the presence of two molecular species. Plant Cell Physiol. 24 (1983) 189-198. Takruri, I.A.H. and Boulter, D.: The amino acid sequence of ferredoxin from Sambacus nigra. Phytochemistry 18 (1979) 1481-1484. Wierenga, R.K., Terpstra, P. and Hol, W.G.J.: Prediction of the occurrence of the ADP-bindin8 ,gap-fold in proteins, using an amino acid fingerprint. J. Mol. Biol. 187 (1986) 101-107. Williams, E. and Bainbridge, B.W.: Genetic transformation in Methylococcus capsulatus. J. Appl. Bacteriol. 34 (197 !) 683-687. Williams, E., Shimmin, M.A. and Bainbridge, B.W.: Mutation in the obligate methylotrophs Methylococcus capsulatusand Methylomonas albus. FEMS Microbiol. Lett. 2 (1977)293-296. Woodland, M.P. and Dalton, H.: Purification and properties of component A of the methane monooxygenase from Methylococeus capsulatug (Bath). J. Biol. Chem. 259 (1984) 53-59. Yamaguchi, M. and Fujisawa, H.: Characterization of NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system from Pseudomonas arvilla C-I. J. Biol. Chem. 253 (1978) 8848-8853. Yubisui, T., Miyata, T., lwanga, S., Tamura, M., Yoshida, S., Takeshita, M. and Nakajima, H.: Amino acid sequence of NADH-cytochrome bs reductase of human erythrocytes. J. Biochem. 96 (1984) 579-582.