Sequence of the Sodium Ion Pump Methylmalonyl-CoA Decarboxylase from Veillonella parvula*. (Received for publication, June 7, 1993, and in revised form, ...
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 268, No. 33, Issue of November 25, pp. 24564-24571,1993 Printed in U.S. A.
Sequence of the Sodium Ion Pump Methylmalonyl-CoA Decarboxylase from Veillonellaparvula* (Received for publication, June 7, 1993, and in revised form, July 23, 1993)
Jon B. Huder and PeterDimrothS From the MikrobiologischesInstitut der Eidgenossischen Technischen Hochschule, ETH-Zentrum, CH-8092Zurich, Switzerland
The genes encoding methylmalonyl-CoA decarbox- boxylases: oxaloacetate decarboxylase and glutaconyl-CoAdeylase fromVeillonellaparvulawere cloned on plasmids carboxylase (1, 2). All these decarboxylases contain a periphusing oligonucleotides derived from N-terminal amino eral membrane-bound subunit of M , 60,000-65,000 that catacid sequences as specific probes. The entireDNA se- alyzes a carboxyl transfer from the substrate to theprosthetic quence of the methylmalonyl-CoA decarboxylase genes biotin group. In methylmalonyl-CoA decarboxylase (3) and together with upstream and downstream regions was glutaconyl-CoA decarboxylase ( 5 ) , the biotin is bound to a determined. The genes encoding subunitsa (mmdA),6 separate biotin carrier protein subunitof apparent M , 18,000( m m d D ) ,t (mmdE), y (mmdC),and B (mmdB) of the 19,000, while oxaloacetate decarboxylase contains the biotin decarboxylase were clustered on the chromosome in on the C-terminal domain of the a-subunit(6, 7). In addition, the given order. The previously unnoted t-chain (Mr all Na’ transport decarboxylases contain a highly hydropho5,888) was clearly shownto be a subunit of the decarbic subunit (@)that migrates as a polypeptide of M , 32,000boxylase by correspondence of the N-terminal amino 33,000 on SDS gels (3,5,8). Thetrue molecular weight of the acid sequence with that deduced from the DNA se&subunit of oxaloacetate decarboxylase from Klebsiella pneuquence of mmdE. The a-subunit was 60% identical with the carboxyltransferase domain of rat liver pro- moniae and Salmonella typhimuriumas derived from the DNA pionyl-CoA carboxylase, the &subunit showed 61% sequence, however, is 45,000 (7). The @-subunitis supposed sequence identity with the &subunit of oxaloacetate to contain several membrane-spanning domains (7, 9). Its function probably is the catalysis of the decarboxylation of decarboxylase from Klebsiella pneumoniae, and the biotin-containingy-subunitwas 29-39% identical the carboxylated biotin carrier andcoupled Na+ translocation with biotin-domains of other biotin enzymes. The 6- across the membrane (1, 2, 8). Oxaloacetate decarboxylase subunit of methylmalonyl-CoA decarboxylase and the containsa third subunit (y) of M , 8,900, which probably y-subunit of oxaloacetate decarboxylase did not show contains one transmembrane a-helix in its N-terminalregion significant sequence homology. The gross structureof and a more hydrophilic C-terminal part that could extend both proteins, however, was similar, consisting of a into the aqueous phase (7, 9). The fourth subunit found in hydrophobic membrane anchor near theN terminus, a methylmalonyl-CoA decarboxylase (3)and glutaconyl-CoA proline/alanine linker, and a remarkable accumulation decarboxylase ( 5 ) ( 6 ) could be related to the oxaloacetate of charged amino acids in the C-terminal part. The decarboxylase y-subunit. The function of this subunit is not sequence of the small t-subunit could be aligned to the yet known. C-terminal region of the &subunit downstream of the The sequence of the N-terminal domain of the oxaloacetate prolinelalanine linker, where the two subunits were decarboxylase a-subunit revealed striking homology to the 5 47% identical. Of considerable interest for themecha- S subunit of transcarboxylase from Propionibacterium shernism of Na+transport are the long stretchesof complete n a n i i that catalyzes the same carboxyl-transfer reaction (6, sequence identity betweenthe hydrophobic &subunits of methylmalonyl-CoA decarboxylase and oxaloacetate 7). The C-terminal biotin-containingdomain strongly resemdecarboxylase and the presence of two conserved as- bled the biotin-containing subunits of transcarboxylase and partic acid residues within putative membrane-span- otherbiotin-containing enzymes. An extended stretch of mostly alanine and proline residues in the N-terminal part of ning helices. the biotin domain could provide the a-subunitwith the flexibility required for the flip-flop movement of the prosthetic biotin group between the catalytic centers of the carboxyltransferase (N-terminal domain of the a-subunit) and the In Veillonella parvula (previously named Veillonella alcaleslyase (probably on the @-subunit).Neither the p- nor the y cens) the free energy of methylmalonyl-CoA decarboxylation subunit of oxaloacetate decarboxylase was homologousto any is conserved by conversion into an electrochemical gradient ofNa’ ions (1, 2). The responsible methylmalonyl-CoA de- known sequence (6, 7). In our efforts to elucidate structure and function of the carboxylase is a membrane-bound biotin-containing Na’ sodium ion transport decarboxylases, we have cloned and pump (3,4). Theenzyme shares a number of properties with sequenced the genes encoding the methylmalonyl-CoA decarthe other members of the family of Na+-transporting decar- boxylase from V.parvula. A comparison of the deduced amino * The costs of publication of this article were defrayed in part by acid sequences with those of the oxaloacetate decarboxylase the payment of page charges. This article must therefore be hereby provides insights into potentially important amino acids, nomarked “aduertisement” in accordance with 18 U.S.C. Section 1734 tably within the membrane-bound @-subunits. solely to indicate this fact. The nucleotide sequencefs)reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank withaccessionnumberfs) L22208. 3 To whom correspondence should be addressed.
EXPERIMENTALPROCEDURES
Materials”. parvula (ATCC 17745) was grown anaerobically on sodium lactate as described (4).Methylmalonyl-CoA decarboxylase
24564
24565
Sequence of Methylmalonyl-CoA Decarboxylase was purified from V. paruula by affinity chromatography on a monomeric avidin-Sepharose column (10). Restriction enzymes were from Boehringer Mannheim (Mannheim, Federal Republic of Germany). Oligonucleotides were custom-synthesized by Microsynth (Windisch, Switzerland). The plasmids pBluescript KS(+) and pBR322 and the host strain Escherichia coli DH5 a were from laboratory stock. Amino Acid Sequence Analysis-The subunits of methylmalonylCoA decarboxylase were separated by SDS-polyacrylamide gel electrophoresis according tothe procedure described in Ref. 11 and electroblotted ontoa hydrophobic polyvinylidene difluoride membrane (Millipore) (12). Blot staining was performed with 0.1% COOmassie Brilliant Blue G250. The filter pieces with the respective subunits were used directly for N-terminal sequence analyses using a protein sequenator (model 470/A; Applied Biosystems) with on-line phenylthiohydantoin-derivative detection using high performance liquid chromatography (model 120; AppliedBiosystems). Cyanogen bromide fragments were prepared by isolating the SDS gel pieces with the respective subunits and treatment overnight with 500 pl of 70% formic acid and 1mg of BrCN at 25 “C. The supernatant was removed and the residual peptides were extracted from the gel 50% formic acid, 25% pieces by treatment with 2 X 0.5mlof acetonitrile, 15% isopropanol, and 10% water for 1 h at 37 “C. The combined supernatant andwash solutions were evaporated in a Speed Vac. The cyanogen bromide fragments were separated by SDS-gel electrophoresis, isolated from the gel, and sequenced. Southern AnalyseslRestriction Map of the mmd Genes-Chromosoma1 DNA of V. paruula was obtained as described (13). The DNA was digested with different restriction enzymes and the fragments were separated by gel electrophoresis in 0.7% agarose (20 X 20 cm). The DNA fragments were blottedontoa Hybond N membrane (Amersham Corp.) and fixed on the matrix by 5 min of irradiation with UV light (320 nm).Restriction fragments were analyzed by Southern hybridization (14) with oligonucleotides 5”CAYGAYAAYATIGA-3’ (oligonucleotidea ) ,5’-ATGAAYAAYTTRAARGT-3’(oligonucleotide y-l), and 5’-GAYGCIATGAAYATG-3’ (oligonucleotide 7-2). Oligonucleotides a and 7-1 werededuced from the Nterminal amino acid sequences of the a- and y-subunit, respectively, and oligonucleotide 7-2 was based on the highly conserved sequence motif Glu-Ala-Met-Lys-Met, which constitutes the biotin binding site of most known biotin enzymes (15). The oligonucleotides were labeled with [y-32P]dATP and used for hybridization as described (16). The information obtained from these experimentsis summarized in the restriction map shown in Fig. 1. Cloning Strategy-For cloning the mmd genes we used the information obtained from the restriction analyses (Fig. 1).Chromosomal DNA (100 pg) wasdigested completely with EcoRI, and therestriction fragments were separated by gel electrophoresis in 0.7% agarose. The desired DNA fragment that hybridized with all three oligonucleotides is about 6.2 kb’ long (Fig. 1).Therefore, DNA in the range of 6.0-6.5 kb was isolated from the gel by electroelution. The DNA was digested with restriction enzymes BamHI and XbaI, which do not cut inside the desired EcoRI fragment but will reduce the number of false EcoRI fragments of the same size. EcoRI fragments of6.0-6.5 kb were subsequently separated from the generated smaller fragments by electrophoresis and isolated from the gel as described above. The isolated EcoRI fragment was further digested with HindIII, which cuts twice inside the EcoRI fragment in sufficient distance to the EcoRI sites so that a “pure” HindIII/HindIII fragment resulted that could be used for cloning part of the mmd genes. Isolated HindIII fragments of2.7 kb were cloned into the pBluescript KS(+) vector and transformed into thehost strain E. coli DH5a (16). The plasmids of 40 recombinant white colonies were isolated (17) and analyzed by hybridization against oligonucleotide 7-1, which yielded 10 positive signals. Cloning of the desired DNA fragment wasverifiedby sequence analysis using oligonucleotide 7-1 as a primer. An amino acid sequence identical to the known N terminus of the y-subunit was derived from the DNA sequence. The plasmid thus obtained was termed pJHl (Fig. 1).Also cloned from the chromosomal DNA were the flanking regions of the pJHl insert. The 2-kb EcoRI/ClaI fragment (pJH2O)was cloned into pBluescript KS(+), and the 3.1-kb NheIIEcoRI fragment (pJH40) was cloned into pBR322 (Fig. 1).Both clones were identified via colony hybridization using the pJH1 insert as a homologous probe. The pJHl insert was DIG-labeled according to the manufacturer (Boehringer Mannheim). DNA Sequence Analysis-The DNA region of the methylmalonylThe abbreviations used are: kb, kilobase(s); ORF, open reading frame.
CoA decarboxylase genes was subcloned from plasmids pJH1, pJH20, and pJH40 and sequenced completely on both strands according to the dideoxynucleotide chain terminationmethod described by Sanger et al. (18). Specific oligonucleotide primers were used to complete the sequence. Computer Analyses-Computer analyses of the DNA and protein sequences were performed with the software package from the Genetics Computer Group of the University of Wisconsin (GCG version 7.1) and the PC-Gene program from IntelliGenetics (Geneva, Switzerland). The program TOP-PRED was obtained from G. von Heijne (Stockholm, Sweden). RESULTS
Cloning of theStructural Genes for Methylmalonyl-CoA Decarboxylase-”terminal amino acid sequence analysis of the methylmalonyl-CoAdecarboxylase subunits was performed in order to synthesizespecific oligonucleotide probes for Southern hybridization and cloning of the methylmalonylCoA decarboxylase genes. The N termini of the p- and the 6subunits were blocked, but those of the a- and the biotincontaining y-subunit could be sequenced and yielded MATVQEKIELL for a andMKKFNVTVNGTAYDVEVNEVKAA for y. Since the DNAsequence reported here is the first one of a Veillonella species, no information of the codon usage was available. Therefore, allpossible codons for agiven amino acid had to be considered in the design of the oligonucleotides. The oligonucleotides synthesized corresponded to the N-terminalsequence of the a-subunit (oligonucleotide a), to the N-terminal sequence of the y-subunit (oligonucleotide y - l ) , and to theconserved biotin binding motif of the ysubunit (oligonucleotide 7-2) (see “ExperimentalProcedures’’). All three oligonucleotides hybridized with the same BamHI, EcoRI, Sad, and XbaI restriction fragments ofV. paruula chromosomalDNA, indicating that at least the genes for the a- and y-subunit form a cluster on the chromosome. Based on the restriction map derived from the hybridization data (Fig. I), the 6.2-kb EcoRIfragment was chosen for cloning the genesfor the a- and y-subunits. However, all attempts toclone this fragmentvia colony hybridization failed due to the high unspecific background signal obtained with the oligonucleotides. Therefore, a new strategy was developed, including an “enrichment”of the 2.7-kb HindIII fragment as described under“Experimental Procedures.” Clone pJH1
1 kbp
-
E
. .’
A P
H
P
c
C
P
P
NK
H”.-P
250 bp -
FIG. 1. Physical map of the V. puruulu genome region encoding methylmalonyl-CoA decarboxylase. The upper part shows a restrictionmap with those enzymes used for cloning the mmd genes on plasmids pJH1, pJH20, and pJH40. The hybridization sites of the oligonucleotides derived from the N termini of the a- and the y-subunit and from the conserved region of the biotinylation site (72oligo) are marked by asterisks. The enlarged map in the lower part delimits the 4.68-kb DNA region sequenced and shows the cluster of the five mmd genes. Restriction sites are abbreviated as follows: A, ApaI; B , BamHI; C, ClaI; E , EcoRI; H, HindIII; K, KpnI; N, NheI; P , PstI; s, SacI, x,XbaI.
24566
Sequence of Methylmalonyl-CoA Decarboxylase
GAATPCTAGAARCTACAAC\AC~~CAGA~;GCCCCTATCGGTAARTTTAWGAP.AMAATGGT
60
GGWGTGATGGTATCCAACACGTTGCATTGCGCG'ITOATRACT
120
GATTTGATGGCTAARGGCG~TCGTATGATT~ACGAAAAACCTCGTTATGG~GCAGGCGGC
180
TCCXAATTGC'MTCTTACATCCAAAAGCAACTGGCGGTGTTTTGCTTGAATTATGTCAA
240
. - 3 5
-
1
0
.
- 3 5 . ACATATMTCCTAGGTTATATATTA4ATATGTATGCTTCTTGAACTTTATTCA"lTATGA . A . . mdA AATAAAATGATACTGTAACAAGTITTATITGEBEE~;CTATAATGGCAACAGTGCAGGAA A
T
V
I
I
L
L
H
E
K
L
A
K
V
K
A
G
G
G
E
E
K
Q
H
A
Q
G
K
M
T
A
R
E
R
L
A
A
K
A
M
L
A
S
K
R
E
N
R
A
P
K
K
H
G
1920 506
N
ATTCCATTATAATTCGATTCGGTFAACATATCATCGAATT~GGGAGATACCT"ICTTTAC P
L
*
. mdD . TACTTTTITAGATGATTATGGAAGGACAAGCAGTTACTACCA~WCTTGGTT
2040 12
M
2100 32
GGAAATCGTACATTTAATCGATCCTACTAAGAAGAAQ~LXGAAGCACCAGCAGCAACTGC E I V H L I D P T K K K K E A P A A T A
2160 52
~CTGTTGCTACTCCAACAGCTACTCCTGT~GCTCCGGCRRA
E
2220 72
Q
2280 92
E
G
Q
A
V
T
T
N
P
W
RATCATGGCGATTAATATGACAGTTGTATT~;C~;TRTTAT
K
R
480 26
F
1980 509
L
M
420 6
L
L
360
E
G T T G A G A A A C ~ A C A T G C T C A ~ G G T A A A A T G ; C ~ ~ C ? T C
V
300
Q
AAAATCGAGTTATTGCACGACTAGCTAGCTAAAGTTAAAGCTGGTGGCGGCG-CGC K
N
I
CGTATGAAATAATACAGTATCCAATT~ACATCATATATATGAGTTGTAGATATACATAT
Y
RACGCTITGGCAATGCTTGCLGTARACGCG-CCG~
I
M
P
540 46
A
V
I
A
N
T
M
P
T
T
V
A
V
T
F
P
A
V
V
A
L
P
I
A
A
N
L
A
G
S
I
A
L
Q
N
GGATGAAGTAGTAGCAGCTATCGTAGGTGCCA~TGGCGATGGGGTACTCATC~AACA D
G A T G A T A A ~ C T T C G T T G A A C T I C A T C A A ~ C G ~ A A A C A ~ G ~ T G T T A A C ~ C600 GGT D D N S F V E L D Q F V K H R C V N F G 66
E
V
V
A
A
I
V
G
A
I
V
A
M
Q
Y
S
S
B
LTTGCATCTATTCGACCTACAGCAACCAGTGCTAARTGGCGC 2340 CAAGAAAAGA~AGAATTACCAGGCGAAGGTGTAGTAACAGGTTATGGTAC~AXGACGGT Q E K K E L P G E G V V T G Y G T I D G
I
660 86
A
S
I
R
P
T
A
T
S
A
K
.
W
R
L
E
G
R
L
S
112
mdE
CGGTAGAGGTTAAAATATTT&~CAWTGTAATGAGCAATGCTACAACAACTAAC CGmAGTATATGCAmGCACAAGATP?CACTGTAGAAGGTGGTTCTCTTGGTGAAATG
R
L
V
Y
A
F
A
Q
D
F
T
V
E
G
G
S
L
G
E
M
720 106
G R G '
(MIS N
T T
A
2400
N
T
115/8
GGTAAAGCTCCATCTCAAGATGTAGTAGCAGTAATCGTTGGTGCATTAGCGGCAATGGGT CATGCTGCTARAATCGTTAARGTACAACGTTTAGCAATGAAAATGGGTGCTCCTATTGTT H A A K I V K V Q R L A M K M G A P I V
780 126
GGTATCAACGATTCCGGCGGTGCTCGTATTCAAGAAGCAGTAGATGCCCTTGC'TGGTTAC G I N D S G G A R I Q E A V D A L A G Y
840 146
GGTAAAATITKmGAAAATACAAAAATGCATCTGGTGTTATTCCACAAATTWCGTAAX G K I F F E N T N A S G V I P Q I S V I
900 166
ATGGGCCCATC;TGCGGGCGGTGC~CTATATTCTCCAGCAT~GACTGAC&ATCTACATG
960 186
G
K
A
P
S
Q
D
V
V
A
V
I
V
G
A
L
A
A
M
2460 28
G
TATTCCGCTGATCAAATCGCGCATATCCGGCAATCGTAAGCTACAA 2520 Y
S
A
D
Q
I
A
H
I
R
P
I
V
S
Y
N
W
K
M
E
48
GGCCGT?TGCGTGGTAATCGAT~GATAAT~GGCTAATTTGATAATCCATGGGA~ACTC G
R
L
R
G
N
R
2580 55
'
AATTTATATGTGTAAAAATATTTATAAGTTTTGTGTAGAAACTACACATTTTGG&GAAT
M
G
P
C
A
G
G
A
V
Y
S
P
A
L
T
D
F
I
Y
M
.
2640
.
mdC
TTAAGATGAAAUATICAACGTTACAGTAAATGGTACAGCATATGATGTAGAAGTTAATG M K K F N V T V N Q T A Y D V B V N E
2700 19
G T T ~ T A C T P C T C A ~ A T C A C T G G W C ~ C A G T A A T C A A R ~ T G T A A C T G G T 1020 206 V K N T S Q M F I T G P A V I K S V T G AAGTGAAAGCAGCGGCXCTGCAGCAGCTCCTARAGCAGC~CAGCAGCAGCTCCAGCTC
GAAGAAGTAACCGCTGAAGAGATCTTGGTGG'ICICAATG E
E
V
T
A
E
D
L
G
G
A
M
A
H
N
S
V
S
G
V
V
1080 226
K
A
A
A
P
A
A
A
P
K
A
A
P
A
A
A
P
A
P
CTAARGCAGCTCC'TGCACCAGCACCAGCXCAGCTGCAGCAGCAGCTCCAGTTCCAGCAG GCTCACTTECAGCTGAAAGATGATTGTAXGCTCAARTTCGTAC~ATTAGGC
A
H
F
A
A
E
N
E
D
D
C
I
A
Q
I
R
Y
L
L
G
F
L
P
S
N
N
M
E
D
A
P
L
V
D
T
G
D
D
P
T
M
1260 286
CGTGAGGATG~GCTTGAATAGCTTGTTGCCAGATAACAGTAACATGCC&ACGACATG R
E
D
E
S
L
N
S
L
L
P
D
N
S
N
M
P
Y
D
~GATGTTATCGCAGCTACTGTAGATAATGGCGAATACTA'TGAAGTAC~CCAWCTAT K D V I A A T V D N G E Y Y E V Q P F Y
A
A
P
A
P
A
P
A
P
A
A
A
A
A
P
V
P
A
G
1320 306
A
E
T
V
K
A
P
M
P
G
K
I
L
S
V
A
V
S
A
G
T
N
I
I
T
C
F
A
R
F
D
G
Q
S
V
G
I
I
2940 99
AAATCGCAGCTCCTCA'TGA'TGCTGTAGTTITCGAAGTTCGCGTATCTGCTAACCAAACTG I A A P H D A V V S E V R V S A N Q T V
3000 119
A
. ~ACCAACC>AATGGC~GGTTGCTIGGACATTAACGCTWTGAC&W~CCGT N Q P K V M A G C L D I N A S D K S S
R
~ATCCG&TGTGATGC~CAATAT?CCAA~~GTTACTGGT
F
I
R
F
C
D
A
F
N
I
P
I
V
N
F
V
D
V
P
G
TTCTTGCCTGGCACAAATCAGWCGGTAXATTCGXATGG'TGCT~TGTTA
F
L
P
G
T
N
Q
E
W
G
G
I
I
R
H
G
A
K
M
L
TA~;~TPAC?~TGAAGCTACAGTACC~AAAATTACTGTTA~CACWGT&GCATATGGC
Y
A
Y
S
E
A
T
V
P
K
I
T
V
I
T
R
K
A
Y
G
1440 346 1500 366 1560 386 1620 406
GG~CTACC~GCTATGTG;TCCCAAGAGTAGGCGCAGACCAAGTATA&C~GCCT G S Y L A M C S Q D L G A D Q V Y A W P
1680 426
;\CAXCGAAR~~GCTGTAATC;GGTCCTGC>GCAGCTA~TATTAW-~AAGAX+GAC T S E I A V M G P A G A A N I I F K K D
1740 446
GARGATAARGATGCTARAAC~GCTAAATATGTAGAAGAGT&GCGACTCC~ACAARGCT A
1800 466
I
1860 486
E
D
K
D
A
K
T
A
K
Y
V
E
E
F
A
T
P
Y
K
GCAGMCGTG~C~GTAGA~TETAAXGAACCTAAAC~AACTCGTCCAGCAGTTATC A
E
R
G
F
V
D
V
V
I
E
P
K
Q
T
R
P
A
V
79
GTCAAGCAGTTEAAAAAGGCGAAACTTTGTTGATWTTGAAGCTATGMTGC~TG Q A V K K G E T L L I L E A M K M Q N E
TATCCACTGGCGATGACATGGTTGTICTTGGCTAATACCAAGTGTTGTATGACGTGGCTG 1380 ~CTACTAATA~CATTACTTGCTTCGCWGT~TEATGGTCLTCCG~GTATCA~CT S T G D D M V V L G ' 326
A
2820 59
GTGCTGAARC~GTARAAGCTCAATGCCTGGT~TCTTATC~ 2880 1200 266
TTC~CCA?~CAACAATAT~GAAGATGC&CATTGGTAGATACTGGTGACGATCCAACT
K
1140 246
2760 39
mdB
. A
3120 5
~GCCACGTCA&TAACAATT"ATATTTGT&~AACGCATATGGAGGCTT~GCT
M
E
A
F
T
T
G
N
A
3180 25
R
E
F
E
P
3240 45
F
3300 65
GTTGCGATACAAWTGTTATGATAGCGGGTTCCWGCA~ACAACAGGCAATGCT
V
A
I
Q
S
V
I
N
D
S
G
F
L
A
F
ATTATGATXTTGTAGGT?TGATCCTATT&ATYTAGCA&CWGTGAGTTCGAACCA I
M
I
L
V
G
L
I
L
L
Y
L
A
F
A
&ATXT~;GGXCGATTGCGTTTGGTIC&ACTTGCCA~TATICCTCGTAACGGTTTC L
L
L
G
P
I
A
F
G
C
L
L
A
N
I
3060 129
P
R
N
G
GAAGAAGGCG~TATGGCGCTTATTAGTGCAGGTAWTCCCL 3360 E
E
G
V
M
A
L
I
S
A
G
I
S
Q
E
I
F
P
P
L
85
~ T T ~ C C T T G G T G T A G G T G C A A ~ ; A C T G A C ~ G T C C A C ~ A ~ C T A A ~ C T W C3 4A2 0 I
F
L
G
V
G
A
M
T
D
F
G
P
L
I
A
N
P
K
T
105
M
3480 125
CTTGOCTTTACAGCTCAAGA~GCGGCTGCTAITGGTATCAGTGGTGG'TGCTGATGGCCCT L G F T A Q E A A A I G I I G G A D G P
3540 145
~CTTTTAGGGGCTGCTGCTCAAAWGGTGTATTTGCTGCCTTAGGTGGCGCAATGATG
L
L
L
G
A
A
A
Q
I
G
V
F
A
A
L
G
G
A
M
ACATCCA~ACTTAGCTACTCTAGCTCTCA~TA~AGGTGC 3600 T
S
I
Y
L
A
T
K
L
A
P
H
L
L
G
A
I
A
V
A
165
FIG.2. Nucleotide and deduced amino acid sequence of methylmalonyl-CoA decarboxylase genes of V.paruula. The nucleotide sequence starts at the EcoRI site upstream of the mmdA gene. The putative 070 E. coli promotors and a possible terminator are ouerlined. Presumptive ribosome binding sites are underlined. Amino acids confirmed by protein sequencing of subunits or peptides are printed in bold letters. The stop codons are marked by asterisks. The lysine residue of the y-subunit that becomes biotinylated (LysS5)is indicated by a bold asterisk.
Sequence of Methylmalonyl-CoA Decarboxylase
24567
ORF2 was unequivocally shown to encode the 6-subunit of the methylmalonyl-CoA decarboxylase by sequencing a BrCN fragment of this subunit. The resulting sequence (Fig. 2) was 3720 ACTCAAAAAGAACGTGAAATCGlTATGGAACAATTGCGCGAAGTAACACGrnAGAAA 205 T Q K E R E I V M E Q L R E V T R F E K identical to amino acids 87-100 of ORF2, which is therefore called mmdD. 3780 ATCGTGTICCCAATCGTTGCAACGATCTTCATTl'CCTTATTGCTKCmCA~ACATCC 225 ORF3 started at position 2377 with an ATG and stopped I V F P I V A T I F I S L L L P S I T S at position 2542 with TAA. This ORF encoded a protein of 3840 CmTAGGTATGTTGATGTGTAACTTGTTCCGTGAATCTGGCGTAACTGATCG~A 55 amino acids with a calculated M, of 5,888. Hitherto, such 245 L L G M L M L G N L F R E S G V T D R L a small protein has never been detected in purified methyl3900 TCCGATACTTCTCAAAATGCATTGATCAATACAGTTACAATCT malonyl-CoA decarboxylase preparations(3). However, as 265 S D T S Q N A L I N T V T I F L A T G T described below, the protein deduced from ORF3 is actually a 3960 fifth subunit of this enzyme and ORF3 is therefore named GGCTTGACAATGAGTGCGGAACACTKTT?AGCTTAGAAACCATCAAAATTATKTTTTA 285 G L T M S A E H F L S L E T I K I I L L mmdE. ORF4 started with an ATG at position 2646 and ended at 4020 GGC'ITATTCGCATTTATITGCGGTACAGCTGGTGGCGTATTGTKGGTAAATTGATGAGC 305 G L F A F I C G T A G G V L F G K L M S position 3033 with a stop codon TAA. The deduced N-terminal amino acid sequence, as well as thepresence of the biotin4080 TTAGTAGATGGTGGTACAAATCCACTTATM3GTCGGTICCA 325 L V D G G K T N P L I G S A G V S A V P binding-site motif (positions 2919-2933), clearly identified ORF4 as the structural gene for the y-subunit, which was 4140 ATGGCAGCTCGCGTATCWAAGTAGTAGGTGCGAAAGCTAACCCAGCTAACTTCTTGCTC therefore termed mmdC. mmdC consists of 129 amino acids 345 M A A R V S Q V V G A K A N P A N F L L with a calculated M, (including the biotin prosthetic group) 4200 A T G C A T G C T A T G G G A C C T A A C G T A T C G G T A A of 12,913. The large discrepancy of this value to theapparent 365 M H A M G P N V A G V I G T A V A A G T M, obtained from SDS-polyacrylamide gel electrophoresis 4260 ATGCTTGCTATGTTGTCCAACCACTAATAATTTAATAATTATTCACCCAGGTGTACCTM (18,500; Ref. 3) probably results from a proline/alanine-rich 373 M L A M L S N H ' sequence between residues 22 and 58. Proteins with proline/ alanine linkers areknown to have reduced mobilities in SDS4320 ATAGGTACACCTATTGGTGAATCAA.W.AGCCC"GGTCATTATGTAATCATACATAAA?A polyacrylamide gel electrophoresis (19). 4380 GGGAATATTKCAATAAATGCTTTKAGATCGGTCATTAGGEATTAGCAAGATTTCACA ORF5 started at position 3106 with ATG and stopped at 4440 GAATAACCTTCTAGTGCTTTAAATATAGTAGAGGTGGTCACTTGTTATCAATTGAATTAA position 4225 with a TAA stop codon. The deduced protein 4500 AAATACTGATITGTITTGTGGCATTTATTGlTITTTTTA'TTACTGCACTGATTATCG consists of 373 amino acids with a calculated M, of38,730. 4560 GECTGAAGGCAAAGCTAAATGGTITTAGCAGAGAACTAAATATTCTTGGT'ITAACCGCC Although not verified by amino acid sequence analysis, the 4620 GAGG"KTTGGTGAAACTTTATAmGGTATCCTAAAACAAGAGAGGGATATGGGA considerable homology to the p-subunit of oxaloacetate de4680 TTAC"TTATAATGGCCTCTGCCATTGGTATCGTAGG?TATAlTl'ATATCTCl"CATAATI carboxylase from K . pneumoniae and s. typhimurium (Fig. 7) FIG.2"continued (7) definitely identified ORF4 as the gene for the p-subunit of methylmalonyl-CoA decarboxylase, and it was therefore could thus be isolated, which encoded the N terminus of the named mmdB. y-subunit as shown by DNA sequencing. From the hybridiBiochemical Verification of a Fifth Subunit of Methylmazation data with oligonucleotide a, it was evident that the 5' lonyl-CoA Decarboxyhe-As described in the previous secend of the gene for the a-subunit was not present on pJH1. tion, DNA sequence analysis led to the discovery of a small Furthermore, DNA sequence analysis of the complete 2.7-kb ORF, located between mmdD and mmdC (Fig. 1).The deduced HindIII insert revealed that the 3' end of the gene for the p- protein consists of 55 amino acids with a calculated M,of subunit was also missing on that fragment. Thus, in order to 5,888. A protein of this size has never been observed in complete the sequences, two additional fragments containing purified preparations of methylmalonyl-CoA decarboxylase the upstream and downstream DNA of the HindIII fragment (3), presumably because of its small size. Therefore, the had to be cloned. For this purpose, the 2-kb EcoRI/ClnI and decarboxylase subunits were separated on a 16.5%rather than the 3.1-kb NheI/EcoRI fragments were chosen and cloned on a 12% SDS polyacrylamide gel. As shown in Fig. 3, a band from chromosomal DNA via colony hybridization using the of about 6 kDa is visible after silver staining but only if large p J H l insert as a homologous probe. The resulting plasmids amounts of protein are loaded on the gel. In order to verify were called pJH2O and pJH40 (Fig. 1). that thisband corresponds to theprotein deduced from ORF3, DNA Sequence Analysis-A DNA regionof 4.68 kb, starting the protein band was eluted from the gel and subjected to Nat theEcoRI site of pJH20, was completely sequenced on both terminal amino acid sequence analysis. In the first cycle, only strands using pJH1,pJH20,pJH40,andappropriate sub- a weak signal was observed (typical for serine), but no signal clones. The complete nucleotide sequence together with the for methionine was found. The following cycles yielded the deduced amino acid sequence is shown in Fig. 2. Five open sequence Asn-Ala-Thr-Thr-Thr, which corresponds to the reading frames (ORFs) were identified, whichwillbe de- deduced N terminus of ORF3. Thus, the existence of a fifth scribed in the order shown in Fig. 1. subunit of the methylmalonyl-CoA decarboxylase was proven. ORFl started at position 403 with an ATG and ended at Apparently, the N-terminal methionine is cleaved off in the position 1930 with a TAA stop codon. Since the deduced N- mature protein, which therefore starts with a serine residue. terminal amino acid sequence of ORFl was identical to the The calculated M, of the mature t-subunit is 5,758. one obtained by protein sequencing of the a-subunit, ORFl Comparison of Amino Acid Sequences of Methylmalonylundoubtedly encodes this subunit and is therefore termed CoA DecarboxylaseSubunits with Those of Other Protein.+" mmdA (mmd is mnemonic for methyl-malonyl-CoA-decar- search of the GenBank and EMBL data banksrevealed relaboxylase). mmdA encodes a proteinof 509 amino acid residues tionships between methylmalonyl-CoA decarboxylase subwith a calculated M, of 55,100. units and other proteins with various degrees of sequence ORF2 started at position 2006 with an ATG and ended at identity. A remarkable degree of 60 or 52% identity was found position 2351 with a TAA stop codon. The deduced peptide between the a-subunit of methylmalonyl-CoA decarboxylase consists of 115 amino acids with a calculated M, of 11,951. and the carboxyltransferase domain of rat liver propionyl-
GCATATTCCTATATGTCCTTGGTACCGTTGATTCAACCACCTGTAATGAAACTCTKACT A Y S Y M S L V P L I Q P P V M K L F T
"
3660 185
Sequence of Methylmalonyl-CoA Decarboxylase
24568
i.e. catalysis of the carboxyl transfer from methylmalonylCoA to protein-bound biotin toyield propionyl-CoA and the carboxybiotin derivative, or catalysis of the carboxyl transfer in the reverse reaction. In contrast, no significant sequence -a homology was found to the carboxyltransferase domain of oxaloacetate decarboxylase (6,7), inaccord with the different substrate specificities of these catalysts. -B No significant sequence homology could be found between the &subunit of methylmalonyl-CoA decarboxylase and the Y 17.1 y-subunit of oxaloacetate decarboxylase (7, 9). The hydro14.5 - P -6 phobicity profile of the two subunits, however, was similar, indicating a hydrophobic and probably membrane-spanning 8.2 N-terminalpartand amorehydrophilic C-terminalpart. Another common feature of the two subunits is a proline/ -E 6.3 1c. alaninelinkerinthe region betweenresidues 47 and 65 (residues 45-60 in case of the oxaloacetate decarboxylase ysubunit). The y-subunit of oxaloacetate decarboxylase consists of 83 amino acid residues and contains, close to the C terminus, a remarkable motive of 4 histidines in series (7, 9), which could provide the binding site for the Zn2+ metal ion present in this enzyme (21). The &subunitof methylmalonylFIG. 3. SDS-polyacrylamide gel electrophoresis of purified CoA decarboxylase is 32 amino acids longer and lacks the may be methylmalonyl-CoA decarboxylase from V . parvula. A step series of multiple histidines. Related to these findings gradient of 5,10, and 16.5% acrylamide was used,and after separation the occurrence of divalent metal ions(Zn2+,Co2+,or Mn2+) in the gel was silver-stained. The five subunits of the methylmalonyl- carboxyltransferase components of biotin enzymes reacting CoA decarboxylase, including the hitherto undetected e-subunit, are on the ketoacid substrates pyruvate and oxaloacetate but not shown in lane 1 . 9 pg of the purified protein were applied to thegel. (e.g. A molecular weight standard is shown in lane M (the corresponding in carboxyltransferases reacting with thioester substrates propionyl-CoA/methylmalonyl-CoA) (15, 22). molecular masses are given in the ordinate). The c-subunit has considerable sequence homology to the C-terminal part (residues 75-117) of the &subunit (Fig. 5), vpmmdA 1 indicating that the c-subunit may have evolved from the 6Rnpccb 1 PStC12 1 subunit by gene duplication of the N-terminal portion. Upstream of the homologous region, the c-subunit has only 15 VpnmdA 30 ~HAQcKMTARERLAKLFDDNSFVELCQFVKHRCVNFG--QEKKELPGEGVVTGYGTIDGR OHKRGKLTARERISLLLDPGSFLESDMFJEHRCADFGMAARGRINGR Rnpccb 60 additional residues with poor homology to the hubunit. QHSPGKQTARERLNNLLDPHSFDNGAFRKHRTPLFGM--DKAWPADGW~RGTILGR pstc12 39 Like other biotin-binding proteins, the y-subunit of meth8 8 LVYAFAODFTVEGGSLGEMHAAKIVKVORLAMKMGAPIVGINDSGGARIQEAVDALAGYG VPmmdA ylmalonyl-CoA decarboxylase contains the biotin-binding lyL W ~ F S ~ D F T V F G G ~ L S G ~ \ H A Q K I C K I M D Q A I T V G A P V I G L ~ S ~ A R I Q E G ~ S L A G Y A 120 Rnpccb PStClZ 97 P V H R A S Q D F ~ G G S A W R D A ~ E G R R D D G T A L L ~ T P F L F F Y D S G G - R I Q E G I D S L S G Y G sine 35 residue upstream of the C terminus within thehighly ................... conserved sequence LEAMKM (15) (Fig. 6). Additional conVpmmdA 148 KIFFENTNASGVIPQISVIMGPCAGGAVYSPALTDFIYMVKNTSQMFITGPAVIKS~E kDa
M
-t -
1
-
....................................... ....................
.............................. ,.*..*, ..*. f . .
Rnpccb Pstc12
180 156
DIFLRNVTASGVIPQISLIMGPCAGGAVYSPALTDFTFMVKDTSYLFITGPEfVKSVTNE KMFFANVKLSGWPQIAIIAGPCACAS-YSPALTDFIIMTKKA-HMFITGPQVIKSVTGE
vpnnndA Rnpccb Pstcl2
208 240 214
EVTAEDLGGAMAHNSVSGVAHFAAENEDDCIAQIRYLLGFLPSNNMEDAPLVDTGDDP~ DV?PEQLGGAKTHTI1ISGVAHRRFDNDVDALCNLREFLNFLPLSNQDPASIRECHDPSDR
VpmdA Rnpccb Pstcl2
268 300
EDESLNSLLPDNSNMPYDMKDVIA4TVDNGEYYEVQPFYATNIITCFARFDGQSVGIIAN LVPELDTWPLESSKA~LDIIHAVIDEREFFEIMP~A~IVIGF~GRTVGIVGN
273
-NTELRDIVPIDGKKGYDVRDVIAKIMWGDYLEVKAGYAT
,
*;,
VpmdD VpmdE
1
MTAPVATPTATPVAPANASAQNEDEWAAIVGAIVGAIVAMGYSSEQIASIRPTATSA~LE MSNATITNGKAPSQDWAVIVGALAAMGYSADQIAHIRPIV-SWKME . . . . . . . . ..** . . .
..*
f...
DVTADELGGAEPIWPSRRIYFVAEDDDI\AELIAKK-LLSFLWNNTEEASFVNPNNDVSP :+..:*** + *::
.....
.............
51
VpmmdD 111 VpmmdD 50
f f .
...................... f
.
f*.f_..
...
.*..fff..f
VpmodA Rnpccb Pstc12
328 360
QPKVMAGCLDINASDKSSRFIRFCDAFNIPIVNFWVPGFLPG~QEGIIRHGA~LY
332
QPSVMSGCLDINASDKAAEFFCDSFNIPLVQL~VPGFLP-V~EYGGIIRHGRKMLY
VPmmdA Rmpccb PstclZ
388 420 391
AYSEATVPKITVITRKAYGGSYLAMCSQDLGAEQVYAWPTSEIAVMGPAGJANIIFKKD-
vpmmdA447 Rnpccb 479 Pstc12 450 VpmdA Rnpccb Pstcl2
501 533 510
...*.
f f f
f
.
f
GRLSGRG GRLRGNR
.
FIG. 5. Amino acid sequence alignment of the 6- and the esubunit from methylmalonyl-CoA decarboxylase ( VprnmdD and VpmmdC, respectively). Symbols for identity and for con-
QPNVASGCLDINSSVKGARNFCDAFSIPLITFVDVPGFLPGTAQEYGGIIRHGAKLLY
servative exchanges are the same as inFig. 4. .................................................. .. vpmdC smbccp StoadA KpoadA 1 PStC13
1 1 461 461
V P d C ** f , smbccp 55 StoadA 516 PKKHGNIPL KpoadA 521 WRKHANVPL 51 A K K P W K L P L L S E E E I ~ E E E K D L M I A T L N K R V A S L E S E L G S L Q S D ~ ~ E D ~ T A I S A Pstc13
55
V p d C Smbccp StoadA 575 KpoadA 581 Pstc13 109
115
AFAEATVPKITVITRKAYGGAYDVMSSKHLLGDTNYAWPTIIFKGH-
*.,,,.._
AYSEATVPKITCLATPTAAP'IWPC-ATVTLVPTPCTPVPSAEIAVMGAEG~IFRKEI
. ... . . . . . . . . . . . . . . . . .
*,,.*f..*,
,
---EDKDAKTAKYVEEFATPYKAAE---RGFWWIEPKQTRPAVINALAMLASKRENPA ---EDVEAAQAEYVEKFANPFPAAV---RGFVDDIIQPSSTPARICCDLEVLASKKVHRP
.......
.........
KAAODPDAMRAEKIEEYQNGSTRR~GQVDDV~DPADTRRKIASALEMYATKRQTRP
..........
.:
. . . .
are given in the text.
CoA carboxylase (20) or the 12S subunit of transcarboxylase from P. shermanii, respectively (15) (Fig. 4). These data are in accord with a commonfunction of these proteins/domains,
.
__
--KAGEGEIPAPLAGTVSKILVKEGI~TVKAGQTVLVLEAMVK
. . . . . f f . . f . . . . . . f.:
*:.
FIG.4. Amino acid sequence alignment of homologous regions in the methylmalonyl-CoA decarboxylase a-subunit ( VpmrndA), in the rat propionyl-CoA carboxylase &subunit (Rnpccb),and in the P. shermanii 12 S subunit of transcarboxylase ( P s t c l 2 ) .Identical amino acid residues in all sequences compared are marked by an asterisk, and conservative exchangesby a dot. Gaps are indicated by lines. References for the sequences used
f
116
.....
.f.ff._,ff
r
f
,
*,
ANQTVSTGDDMWLG SGQTVDAGDNLITIA SGDAVSVGDTLMTLA AGDAVAVGDTLMTEA ERDAVQGGQGLIKIG
. . . . * . . . . .. FIG. 6. Comparison of the amino acid sequences of biotin
enzymes in the region of the biotinylated lysin with that of the y-subunit of methylmalonyl-CoA decarboxylase (VpmmdC).Smbccp, biotin carboxyl carrier protein from S. mutans; StoadA, a-subunit of oxaloacetate decarboxylase from S. typhimurium; KpoadB, a-subunit of oxaloacetate decarboxylase fromK. pneumoniae; Pstcl3, 1.3 S subunit of the transcarboxylase from P. sher-
manii. Symbols for identity and for conservative exchanges are the same as in Fig. 4. References are given in the text.
Sequence of Methylmalonyl-CoA Decarboxylase
24569
1
served amino acids are scattered over the entire C-terminal x P-subunit region following the Conserved&subunit alanine/proline pairat position 65/66, which is required for recognition by biotin ligase (23). A remarkable sequence of the methylmalonyl-CoA decarboxylase y-subunit is a proline/alanine linker between residues 22 and 58, which contains 23 alanine, 11proline, 2 lysine, and 1 valine residue. The oxaloacetate decarboxylasesfrom S. typhimurium andK. penumoniae also contain proline/alanine linkers, which start 15 residues more downstream than that of methylmalonyl-CoA decarboxylase and extend to the same C-terminal position (6, 7). The biotin carrier proteinof transcarboxylase of P. shermanii contains an accumulation of 9 glycine, 6 alanine, and 2 prolines out of 19 residues in this -- - - region (15), and the biotin carrier of Streptococcus mutuns J 2has 4 glutamines, 2 prolines, and3 alanines in a stretch of 14 100 200 300 0 100 0 amino acid residues (possible Q-linker; Ref. 24) at the same lrsidue numbers location. All these amino acid sequences are supposed to serve the same purpose, i.e. to provide the protein with flexibility FIG. 8. Hydropathy plot of the 0- and the &subunit from for movement of the prosthetic biotin group betweendifferent methylmalonyl-CoA decarboxylase. The hydrophobicity value catalytic centers on the respective biotin-containing enzyme was calculated with the program TOP-PRET (29). Bars show the position of the predicted integral membrane helices. complexes. The N-terminal portion of the methylmalonyl-CoA decarboxylase y-subunits (residues 3-17) is highly homologous to and residues 12-15 of the methylmalonyl-CoA decarboxylase the biotin-carrier protein of transcarboxylase containing 12 @-subunit are an insertion with respect to the oxaloacetate identical residues and 3 conservative exchanges. This region decarboxylase @-subunit.Of interest areseveral longstretches of the transcarboxylase subunit is known to be essential for of amino acid sequence identity between the @-subunits of methylmalonyl-CoA decarboxylase and two different oxaloacbinding to the 12 S carboxyltransferase subunit (25). The degree of sequence identity between the two biotin-carrier etate decarboxylases, which indicate functionally important proteins, as well as between the 12 S subunit of transcarbox- regions of these proteins. We have recently taken advantage ylase and the a-subunit of methylmalonyl-CoA decarboxylase, of these highly conserved areas for amplifyingpart of the gene indicates a similar binding between carboxyltransferase and for the @-subunitof methylmalonyl-CoA decarboxylase from Propionigenium modestum by the polymerase chain reaction biotin carrier protein subunits in bothcomplexes. The amino acid sequences of the @-subunits from methyl- technique.' The hydrophobicity plotof the @-subunit(Fig. 8) malonyl-CoA decarboxylase and oxaloacetate decarboxylase indicates a very hydrophobic protein with several putative are 61% identical (7) (Fig. 7). The homology extends over the membrane-spanning a-helices. Depending on the computer entire sequence with the exception of a long deletion of 57 programs used, secondary structuremodels with 8-11 helices amino acids in the methylmalonyl-CoA decarboxylase @-sub- can be predicted. As the @-subunit secondary structure is unit following residue 64. Additionaldeletions of 1 and 6 probably conserved, the model should also fit the dataderived amino acids are found after residue 73 and 124, respectively, from the oxaloacetatedecarboxylase sequences (7). We therefore prefer a secondary structural model for the P-subunit with 9 transmembrane a-helices. V p d B 1 MEAFAVAIQSVINDSGFLAF~NAIMILVGLILLYLAFAREFEPLLLGPIAFGCLL~I "
KpoadB StoadB
1 1
VpmdB KpoadB StoadB 57
61 57
**,..,.)_..,.**
MESLNALIQGL----GLMHLGAGQAIMLLVSLLLLWLAIAKKFEPLLLLPIGFGGLLSNI
MESLNALLQGM----GLMHLGAGQAIMLLVSLLLLWLAIAKKFEPLLLLPIGFGGLLSNI ._..*_** f f f f. * f f f
*+.... .*..
I . .
. .
...
DISCUSSION
pRNG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nucleotide Sequence-We show here thatthe mmdA, mmdD, mmdE, mmdC, and mmdB genes encoding subunits V p d B 6 5 - F E E G V M A L I - S A G I S Q E I F P P L I F L G V G ~ F G P L I A N P K T L L L G ~ Q I G ~ ~ Ga G , 6, t, y, and @, respectively, of methylmalonyl-CoA decarKpoadB 117 GYSAGVLAIFYKVAIGSGIAPLVIFMGVG~FGPLLANPRTLLLGAALG StOadB 117 boxylase are clustered on the genome of V. purvulu in the GYTPGVLALFYKVAIGSGVAPLVIFMGVGAMTDFGPLLALG given order. Putative ribosome binding sites are located upVpmndB 123 A"L~~~~--GFTAQEAARIGIIGGADGPTSIYLATKLAPHLLGAIAVAAYSYMSLVPLI KpoadB 177 stream of each gene(Fig. 2). Several putative E. coli 070 ALTLNYFGIISFTLPQAAAIGIIGG~PTAIYLSGKLAPELLGAIAVAAYS~LVPLI StoadB 177 ALTLNYFGLISFTLPQAAAIGIIGGADGPTAIYLSGKLAPELLGAIAVAAYS~LVPLI promotor consensussequences (consisting of the -35 and -10 *. regions) are detectable within400 base pairs upstream of the VpmndB 177 QPPVMKLF?TQKEREIVMEQLREVTRFEKIVFPIVATIFISLLLPSITSLLGMLMLGNLF KpoadB 237 QPPIMKALTTDKERKIRMVQLRTVSmEKILFPAVLLLLVALLLPDAAPLLGMFCFGm mmdA gene (two of these are indicated in Fig. 2 by overlining). StoadB 237 QPPIMKALTSETERKIRMVQLRTVSKREKILFPWLLMLVALLLPDAAPLLGMFCFGNLM *.*_** ,.,_,.*_* , , __ __***., . . The upstream region also contained the 3'-part of an open VDmndB 237 RESGVTDRLSDTSQNALINTTIFLA~~LTNSAEHFLSLETIKIILLGLFAFICGTAG reading frame (residues 1-250 of the sequenced part of the KboadB 297 RESGWERLSDTVQNALINIVTIFLGLSVGAKLVADKFLQPQTLGILVLGVIAFCVGTAA StoadB 297 RESGWERLSDTVQNGLINIVTIFLGLSVGAKLVADKFLQPQTLGILLLGVIAFGIGTAA genome). The derived amino acid sequence was not homolo.*fff **(...,..**., *,;* *,(.*(,.* gous to any protein in the data base. VpmdB 297 GVLFGKLMSLVDGGKTWPLIGSAGVSAVPMAAEVSQWGAKANP?LNFLL"GPNVAGV KpoadB 357 GVLMAKLMNVFSRHKINPLIGSAGVSAVPMAARVSNKVGLEACSQNFLLMHAMGPNVAGV 2 x 8 nucleotides, 32 base pairs A palindromic sequence of St'a?.de 357 GVLMAKLLNLCSKM(INPL1GSAGVSAVPMAAEVSNKVGLESDAQNFLL"lGPNVAGV fff__*f._. . _ _ _ ****.*..**..**. downstream of the stopcodon of the @-subunit, that might be 357IGTAVAAGTNLAMLSNH VpmndB functioning as a terminator is indicated in Fig. 2 by overlining. 417IGSAIAAGVMLKWLAM KpOadB StoadB 417IGSAIAAGVMLKWLAM An open reading frame in this downstream region starts at , position 4422 with TTG and extends to the end of the seFIG. 7. Amino acid sequence alignment of the &subunits from methylmalonyl-CoA decarboxylase ( VpmmdB) and ox- quence. No homology of the deduced protein sequence to any aloacetate decarboxylase from K . pneumoniae (KpoadB)and known protein sequence could be found. A translation of this S. typhimurium (StoadB).Symbols for identity and for conserv- ORF into protein seems unlikely because more than 21% of PEAGLALTALESLLAHRDPAQLAVIAAKLHCAPDVHAIKAM PEAGLALTALESLLAHHDAGQLAVIAAKLHCAPDVHAIKEM f
_
_ ,
f
Ilf,.,,
_,;,
, _
f
f.,.*.*.*.ffff_*f.
.*
_,If.,.f.f.fff*f_f.~,_.f*f
f
f
f f * _ * , ,
*e*.*
, , f
f,*.f.f.f*..f.f.fffff_
f f ( I , f f f
f f
* * f , f .
f
_ ,
f f
f f f * t . t t t ( * , . * ~ , f
*f.**f.*fff.f_ff*ft
ff..,
t f f
_ I ,
f t f
,
,
ative exchanges are the same as in Fig. 4. References are given in the text.
* P. Burda, unpublished observation.
24570
Sequence of Methylmalonyl-CoA Decarboxylase
the amino acids would be aromatic. In addition, the G+C origin of this subunitby gene duplication, a correlation of the content in the region of the ORF is 10% lower (-32%) than e-subunit with function is presently not available. the G+C content of the mmdgenes and the ORF found Of special interest for the mechanismof Na' translocation upstream of the a-subunit, and TTG is a rarely used start is the structure of the P-subunit.A secondary structuralmodel codon in Gram-negative bacteria. is shown in Fig. 9. It is based on predictionsmade by hydroOur attempts to clone all mmd genes together in a high phobicity analysis under consideration of the positive-inside copy number plasmid in E. coli have not been successful, rule(29)(Fig. 8 ) andalsotakesintoaccountarguments possibly becauseexpression of methylmalonyl-CoA decarbox- discussed previously for postulating the secondary structure ylase is lethal to anE. coli cell. The enzyme also catalyzes the of the P-subunitof oxaloacetate decarboxylase from K. pneudecarboxylation of malonyl-CoA (3) and will thus interfere moniae and S. typhzrnuriurn (7). It is assumed that the strucwith fatty acid biosynthesis. It is interesting that the genes tures of the different P-subunits were conserved during evoencoding methylmalonyl-CoA decarboxylaseand those encod- lution. TheN terminus is supposed to reach into the periplasm ing oxaloacetate decarboxylase are clustered on the chromo- because it contains 2 negatively charged and no positively some and that the genes encoding the P-subunits of both charged residues, while the end of the first transmembrane enzymes aretranscribedlast (7, 9). In contrast, the gene helix (residues 17-40) of the ,&subunit of methylmalonyl-CoA encoding the a-subunitof glutaconyl-CoA decarboxylase from decarboxylase contains an arginine and that of oxaloacetate Acidaminococcus ferrnentans is separated on the genome from decarboxylase contains 2 lysines sideby side. These positively the genes encoding the additional subunits of the decarbox- charged aminoacids are supposed to functionas stop transfer signals (30). In this firsthelix 12 residues are identicalin the ylase (26). three different p-subunits and the others are conservative The Protein-Biochemicalevidence has indicated structural and functional relationships among the biotin-contain- exchanges. It follows another highly conserved area of 19 ing Na+-transporting decarboxylases oxaloacetate decarbox- amino acid residues containing 7 hydrophobic residues.In our ylase,methylmalonyl-CoAdecarboxylase, and glutaconyl- model, this region was not postulated to traverse the membrane, because the putative helix would be rather short and/ CoA decarboxylase (1, 2). Theserelationshipshavebeen refined by the complete primary structure of methylmalonyl- or would contain a charged glutamate residue. Nevertheless, it CoA decarboxylase reported here. All these decarboxylase the highly conserved sequence of this area indicates that is Veillonella P-subunit there then complexes consist of a tightly membrane-bound @-subunit, functionally important. In the follows a large gap (Fig. 5 ) . T h e second membrane helix was which contains a bindingsite for Na' (8, 27, 28) andis therefore most likely responsible for Na+ translocation across postulated to run from Ilea' to Ala'". A short loop of three the membrane. The more than 60% identity of the aminoacid amino acids connects helix I1 with helix 111, which runs from sequencesbetweenmethylmalonyl-CoAdecarboxylase and Thrlo5 toLeu126.After a short loop the polypeptide traverses the membrane again (Alai33to T h P ) . T h econserved amino oxaloacetate decarboxylase @-subunits is clear evidence for provide the linkage to helix the same function of these proteins in each of these enzyme acids Lys-Leu-Ala-Pro-His-(Glu) ~ ~loop . with many charged residues complexes. The decarboxylases also contain a peripheral a- V, which ends withG ~ u ' A subunit actingas carboxyltransferase. The sequences of these leads t o helix VI (IleZw to Gly233),which is not as highly carboxyltransferases are homologous to those of biotin-con- conserved as the other putative membrane-spanning a-helitaining carboxylases or transcarboxylase with the same sub- ices. This region of the protein is highly hydrophobic in all middle of strate specificity. Transcarboxylase in fact contains two car- theP-subunits sequenced.An aspartateinthe boxyltransferases ( 5 and 1 2 S subunits) withsequence ho- putative helix VI in oxaloacetate decarboxylase, however, is mology to the a-subunit of oxaloacetate decarboxylase and replaced by serine in the methylmalonyl-CoA decarboxylase (15). The sequence. It follows another region with many charged amino methylmalonyl-CoAdecarboxylase,respectively biotin carrier protein isa distinct domainof the oxaloacetate acids that is certainly not integratedin the lipid phase and a decarboxylase a-subunit (6,7) but occurs as a separate protein more hydrophobic region that could coil into helix VI1 to Ala271).Interestingly, the putative helix VI1 of both oxalomoiety in the decarboxylases acting on thioester substrates (3, 5). It is interesting that these biotin binding subunits @"PMAA containalanine/prolinelinkersintheirN-terminal region m a v that are farmore extended than putative linkersequences in the biotin carriersof carboxylases and transcarboxylase (6,7, 15).The reason for thesedifferences may be a requirement in the decarboxylasesfora more extended movement of the prosthetic biotin group between the two catalytic centers of the carboxyltransferase and thelyase (decarboxylase). The6subunit of methylmalonyl-CoAdecarboxylase seems to be anchored in the membraneby a transmembrane helix in the N-terminal region. Following this,theproteincontains a proline/alanine linker, a very hydrophilic strech of 9 amino acids (containing 2 asparagine, 2 glutamines, and 3 aspartates), a hydrophobic region of 12 amino acids, and a hydrophilic C-terminal tail. No sequence homology of this protein was found to the y-subunitof oxaloacetate decarboxylase (7, FIG. 9. Secondary structure model of the membrane-bound 9), but the predicted secondary structures were surprisingly similar. Whether this similarityreflects a functional relation- &subunit of methylmalonyl-CoA decarboxylase. The boxes show the putative transmembrane regions of the polypeptide. Basic ship is presently unknown. Remarkable is the presence in residues are boxed, and acidic residues are indicated by circles. Amino methylmalonyl-CoA decarboxylase of a small polypeptide (Mr acid residues conserved in the &subunits of oxaloacetate decarbox5,758, t-subunit) thatshows strong sequence homology to the ylase from K. pneumoniae and S. typhimurium are in bold letters. The C-terminal portion of the &-subunit. While this indicates an top part represents the periplasm.
Sequence of Methylmalonyl-CoA Decarboxylase acetate decarboxylase @-subunits contains alysineresidue, which is replaced by threonine (position 268) in the methylmalonyl-CoA decarboxylase sequence. It is conceivable that the non-conserved charged amino acids in putative helices VI and VI1 of the oxaloacetate decarboxylase P-subunit form a salt bridge within the membrane. The next helix (VIII) is rather clearly defined by its strong hydrophobicity (Ile2s2t o Gly301).The following region contains one of the most highly conserved areas of the whole protein (Asn313t o Ser331),where all 19 amino acid residues are identical. This area is flanked on both sides by less conserved segments with a number of charged residues. We do not want to speculate whether the mainlyhydrophobicconserved region traversesthemembrane. If so, an arginine would be located within the membrane-bound part. The polypeptide chain could traverse the membrane again(helix IX) fromPhe343 to leaving the short C-terminal peptide Ala-Met-Leu-Ser-Glu-His extending into the aqueous phase. Most of the helices predicted by this model are uncharged. Aconserved aspartate residue isfoundinthe middle of putative helix I1 and helix IV in highly conserved areas of the protein. These aspartic acids may be important residues for Na' translocation. As discussed above, the proposed model contains a number of disputable elements that must in the future be investigated by topological studies.
1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Acknowledgments-We thank Dr. Rocco Falchetto (Proteinchemie Service Labor of the Eidgenossischen Technischen Hochschule Zurich) for the performance of protein sequencing, Patricie Burda for participation in part of the subcloning and sequencing, and Dr. Michael Bott for improvement of the style of the manuscript.
26. 27.
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