Báo cáo Y học: Propionate CoA-transferase from Clostridium propionicum Cloning of the gene and identi®cation of glutamate 324 at the active site pdf

Propionate CoA-transferase from Clostridium propionicum Cloning of the gene and identi®cation of glutamate 324 at the active site Thorsten Selmer, Angela Willanzheimer and Marc Hetzel FB Biologie, Philipps-Universita È t, Marburg, Germany Propionate CoA-transferase from Clostridium propionicum has been puri®ed and the gene encoding the enzyme has been cloned and sequenced. The enzyme was rapidly and irre- versibly inactivated by sodium borohydride o r h ydroxyl- amine in the presence of propionyl-CoA. The reduction of the t hiol ester between a catalytic site glutamate and Co A with borohydride and the cleavage by hydroxylamine were used to introduce a site-speci®c label, which was followed by MALDI-TOF-MS. This allowed the identi®cation of glutamate 324 at the active site. Propionate CoA-transferase and similar proteins deduced from the genomes of Escherichia c oli, Staphylococcus aureus, Bacillus halodurans and Aeropyrum pernix are proposed to form a novel subclass of CoA-transferases. Secondary structure element predic- tions were generated and compared to known crystal structures in the d atabases. A h igh degree of structural similarity w as obse rved between the arrange ment o f s ec- ondary structure elements i n these proteins and glutaconate CoA-transferase f rom Acid aminoc occus fermentans. Keywords: Clostridium propionicum; alanine metabolism; CoA-transferase; active site; thiol ester. Clostridium propionicum has been isolated as an alanine fermenting organism from the black mud of San Francisco bay [ 1]. Th e fermentation products were acetate, ammonia, carbon dioxide and propionate [2]. In contrast to other organisms, which ferment alanine according to the so-called randomising pathway with succinate a s a symmet- ric intermediate, C. propionicum ferments alanine via the nonrandomising pathway with acrylyl-CoA as c haracter- istic intermediate. This pathway seems to be restricted to a limited number of o rganisms, including Megasphaera elsdenii [3], Bacteroides ruminic ola [4], C. propionicum and Clostridium homopropionicum [5]. Serine and cysteine are fermentedinasimilarmannerbyC. propionicum yielding acetate and propionate. Likewise, threonine is fermented to propionate and butyrate as volatile fatty acid end products. As outline d in Fig. 1, alanine undergoes an initial oxidative cleavage to a mmonia and pyruvate, the latt er being either oxidized to carbon dioxide and acetate o r reduced to (R)-lactate. (R)-Lactate is subsequently reduced to propionate by reactions carried out at the c oenzyme A thiol ester level rather than using the free carboxylates. A critical step in the reductive branch of this pathway is the activation of (R)-lactate as its (R)-lactoyl-CoA derivative. This reaction is carried out by the enzyme propionate:ace- tyl-CoA CoA-transferase (EC 2 .8.3.1, also known as pro- pionate CoA-transferase), which has b een demonstrated to activate (R)-lactate using the end product of the reduction, propionyl-CoA, or acetyl-Co A as a c oenzyme A donor [6] (Eqn 1). RÀlactate  propionyl À CoA  propionate RÀlactoyl À CoA 1 The enzyme has been p reviously puri®ed and character- ized as a homotetrameric enzyme (a 4 ) with an apparent molecular subunit m ass o f 6 7 kDa [6]. Although a prefer- ence of (R)- lactate over (S)-lactate was observed, the enzyme exhibited a rather broad substrate speci®city for monocarboxylic acids including acrylate, propionate and butyrate whereas dicarboxylic acids were not used. The g eneral mechanism for the C oA-transferases has been suggested to proceed via the successive formation o f a m ixed anhydride between the C oA-donor carboxylic acid an d an essential glutamate residue of the enzyme, followed by t he formation of an e nzyme-CoA thiol ester intermediate. The product is then f ormed by a n inverted sequence of these steps with the acceptor carboxylate [7]. More recently, a number of CoA-transferases have been discovered, which apparently do not follow this general mechanism [8,9]. Formation of an enzyme-CoA thiol ester can be detected either by site-speci®c cleavage of the polypeptide chain [10], reduction of the t hiol ester w ith sodium borohydride [11±13] or directly by mass spect- rometry [14]. Alternatively the mixed anhydride interme- diates can be detected by the formation of stable derivatives of the catalytic glutamate and hydroxamic acids [15] or indirectly by oxygen exchange experiments [14,16]. To date, only the crystal structure of glutaconate CoA- transferase (EC 2.8.3.12) from Acidaminococcus fermentans has been solved. In contrast to the homotetrameric propi- onate CoA-transferase of C. propionicum,theformer enzyme is a hetero-octameric protein (a 4 b 4 ), whose structure belongs t o t he open a he lix/b sheet protein family and has Correspondence to T. Selmer, FB Biologie, Philipps-Universita È t, D-35032 Marburg, Germany. Fax: + 4 9 6421 2828979, Tel.: + 49 6421 2825606, E-mail: selmer@mailer.uni-marburg.de Enzymes: propionate CoA-transferase (EC 2.8.3.1); glutaconate CoA-transferase (EC 2.8.3.12). (Received 3 August 2 001, revised 5 November 2001, accepted 7 November 2001) Eur. J. Biochem. 269, 372±380 (2002) Ó FEBS 2002 been described as four-layered a helix/a helix/b she et/a helix type with a novel topology [17]. T his topology is found in both subunits and differs considerably from other a helix/b sheet proteins including nucleotide-binding domains. In this communication, we report the cloning and sequencing of the gene encoding propionate CoA-transfer- ase from C. prop ionicum and present experimental evidence that glutamate 324 acts as the catalytic carboxylate. MATERIALS AND METHODS Materials Sequencing grade proteases were pur chased from Boehrin- ger Mannheim (Germany). Coenzyme A (trilithium salt) was from ICN Biomedicals (Eschwege, Germany). All other chemicals were of the highest grade available and from common commercial sources. C. propionicum (DSMZ 1682) was purchased from the German collection of microorganisms and cell cultures (DSMZ, Braunsc hweig, Germany). Synthesis of acyl-CoA substrates Acetyl-, butyryl- and propionyl-CoA were prepared from the corresponding anhyd rides and CoA by the method of Simon & Shemin [18]. All CoA-derivatives were puri®ed as described previously [14]. Enzyme assay The enzyme test for propionate CoA-transferase activity was carried out at 25 °C as described previously [19]. Cultivation and storage of microorganisms C. pr opionicum was cultivated in a complex medium containing D , L -alanine as the s ole s ource of energy, as described previously [6]. Freshly prepared anaerobic media were inoculated with 5 to 20% stationary or late exponential precultures and grown for 24 to 36 h at 37 °C. The cells were harvested by centrifugation and stored at )80 °C. Preparation of cell free extracts of C. propionicum Frozen cells (20 g) were suspended i n 100 mL of 25 m M potassium ph osphate, 1 m M dithiothreitol, 1 m M EDTA, 1m M MgCl 2 , pH 7.0 (Buffer A). The suspension was homogenized by sonication on ice for 15 min. Poly(eth yl- eneimine) (0.2% m/v ®nal concentration) was added and the crude extract w as centrifuged for 45 min at 100 000 g.The clear supernatant was stored on ice until used. Puri®cation of propionate CoA-transferase All puri®cation steps were carried out at 4 °C. The clear extract was applied to a Q-Sepharoseä column (2.6 ´ 10 cm, Pharmacia) equilibrated with buffer A. The column was w ashed with 50 mL o f buffer A and developed bya500-mLlineargradientof0to500m M NaCl in b uffer A. Fractions containing CoA-transferase activity w ere pooled and adjusted t o a ®nal concentration o f 1 M (NH 4 ) 2 SO 4 11 . The solution was centrifuged for 45 m in at 100 000 g and app lied i n four aliquots to a Resource-Pheä column (1 mL vol., Pharmacia) equilibrated with 1 M (NH 4 ) 2 SO 4 in buffer A. The column was washed with 5 m L of the starting buffer and the proteins were eluted in a 50-mL linear gradient from 1 to 0 M (NH 4 ) 2 SO 4 .The pooled enzyme was dialysed overnight against 5 m M of each boric, citric and phosphoric acids and 5 m M Tris adjusted to pH 7.0 with KOH (buffer B) and then applied on a Resource-Qä column (1 mL vol., Pharmacia) equilibrated with buffer B. T he protein w as eluted by a linear gradient (50 m L) from 0% to 100% of buffer B adjusted to pH 2.0 with HCl. The collected fractions at pH 5.0±5.1 were immediately neutralized by the addition of potassium phosphate, pH 7.5. When required, the protein solution was adjusted t o a ®nal concentration of 200 m M sodium acetate and subjected to gel ®ltration o n S ephadex G 25, equilibrated with buffer A, t o obtain CoA-free protein. T he puri®ed enzyme was ®lter-sterilized and stored a t 4 °Cfor several months without signi®cant loss in activity. The purity of the enzyme was con®rmed by SDS/PAGE with Coomassie-staining of the proteins and by RP-HPLC followed by mass spectrometry [14]. Fig. 1. The fermentation o f alanine by Clostridium propionicum. Th e amino acid alanine is fermented according to Eqn (1) alanine + 2 H 2 0 ® 3NH 4 + +CO 2 + aceta te ± + 2 propionat e ± , DG°  )135 kJ ámol )1 acetate. The enzymes in volved are p yru- vate:glutamate transaminase ( 1), g lutamate dehydrogenase (2) (R)-lactate dehydrogenase (3), propionate C oA-transferase (4) (R)lactoyl-CoA dehydratase (5), acrylyl-CoA reduc tase complex (6), pyruvate:formate lyase (7), pho sphotransacetylase (8), acetate kinase (9) and formate dehydrogenase (10). 2OG, 2-oxoglutarate. Ac-CoA, acetyl-CoA. Ac-P i , acetylphosphate. Ó FEBS 2002 Propionate CoA-transferase (Eur. J. Biochem. 269) 373 N-Terminal sequencing Puri®ed e nzyme (20 lg) was subjected to SDS/PAGE and blotted onto a poly(vinylidene di¯uoride) m embrane. A fter staining of the membrane w ith Coomassie blue R 450, the protein band was cut out and subjected t o N -terminal sequencing by gas-phase Edman degradation. Generation and puri®cation of internal peptides The p uri®ed enzyme (20 0 lg) was freeze-dried, reductively carboxymethylated and digested with trypsin as described previously [14]. T he peptides were puri®ed by RP-HPLC using a Supelco sil-LC318 column (4.6 ´ 250 mm, 5 lm, 300 A Ê ) equilibrated with 0.1% ( v/v) tri¯uoroacetic acid and eluted with a line ar gradient of 0±42% (v/v) acetonitrile within 42 min. The elution of the peptides was monitored simultaneously at 210 and 280 nm. Peptides exhibiting an absorbance at 280 nm were re-applied to the same column using an identical gradient in the presence of 0.1% (v/v) hexa¯uoroacetone-ammonia, pH 6.0, and analysed by MALDI-TOF-MS as described previously [14]. Two pep- tides of suitable size and purity were subjected to N-terminal sequencing. Cloning and DNA-sequencing A degenerated primer pair was deduced from the N-terminus of the enzyme a nd from an in ternal peptide (Table 2) and used to amplify a % 300-bp PCR p roduct from genomic DNA of C. propionicum. T he PCR p roduct was cloned and sequenced using the TOPO TA-cloning kit according to the manufacturer's instructions. The PCR-product w as labelled with digoxygenin a nd used to screen EcoR1 fragments of genomic DNA from C. propionicum cloned in a k-ZAP-Express phage. Two positive clones were isolated, plasmids were excised from the vector and the inserts were sequenced using standard laboratory protocols. The sequence data w ere analysed using the EXPASY (Expert Protein Analysis System) server of the Swiss Institute of Bioinformatics, the c omputational molecular biology facilities provided by the National Institute of H ealth and the 3 D - PSSM Web Server V 2.0 provided by the I mperial Cancer Research Fold Re cogni- tion Server [20,21]. Site-speci®c label of the catalytic glutamate residue Propionate CoA-transferase (50 lg) was incubated for 2 m in at 25 °Cin50m M potassium phosphate, pH 7.0, either in the presence o r absence of 100 l M propionyl- CoA. Either hydroxylamine hydrochloride (pH 7.5, 200 m M ®nal concentration) or sodium borohydride (20 m M ®nal concentration) were added and the reaction was a llowed t o proceed for another 10 min at 25 °C. Aliquots of t he samples were assayed for CoA-transferase activity. The samples were reductively carboxymethylated andthebufferwasexchangedbygel®ltrationtoyield 50 m M ammonium acetate, pH 8.0, 10% (v/v) acetonitrile. Each sample was s plit into four equal volumes and digested for 16 h at 37 °C i n the presence of either 2% (w/w) chymotrypsin, endoproteinase AspN, endoprotein- ase GluC ( V8 protease) or 2% ( w/w) trypsin. The samples were acidi®ed with 0.01 vo l. of 2 M tri¯uoroacetic acid and analysed by MALDI-TOF-MS. RESULTS Puri®cation of propionate CoA-transferase The propionate CoA-transferase was puri®ed from C. pro- pionicum grown on a lanine as the sole s ource of energy and carbon. Ion exchange chromatography on Q-Sepharose completely separated t he propionate CoA-transferase (elu- tion at 270±285 m M NaCl) from phosphotrans-acetylase (elution at 130±150 m M NaCl), which contributed to 50% of the apparent transferase activity in cell-free extracts. Most of the contaminating proteins w ere removed by hydrophobic interaction chromatography on a Resource- Phe column [elution at 700 m M (NH 4 ) 2 SO 4 ]. The remaining impurities were removed b y ion exchange chromatography on Resource-Q u sing a d ecreasing pH-gradient (elution at pH 5.1±5.0). As demonstrated in Fig. 2 and Table 1, t he enzyme was essentially pure after these puri®cation steps. The enzyme was 37-fold enriched to a speci®c activity of 85 U ámg pro- tein )1 . In addition to the predominating polypeptide with an apparent molecular mass of 60 kDa in SDS/PAGE, t wo faint bands were observed around 40 and 20 kDa. These additional bands were completely absent when the puri®ed enzyme was incubated in t he presence of sodium acetate followed by gel ®ltration on Sephadex G25, but accounted for up to 30% of the total protein when the enzyme was incubated with 100 l M propionyl-CoA, prior to sample preparation. These data indicated that a small but signi- ®cant fraction of the puri®ed CoA-transferase w as trapped as the enzyme-CoA thiol ester intermediate. It has been Fig. 2. The p uri®cation of propionate CoA-tran sferase. ACoomassie blue staine d 10% SDS/PAGE is shown. The arrows indicate the position of two faint bands at 40 kDa and 20 kDa in lanes 2±4 which are absent in lane 5. Cell free extract (lane 1, 50 lg), Q-Sepharose (lane 2, 10 lg), Resource-Phe (lane 3, 5 lg) , Resource-Q (lane 4, 5 lg). The protein in lane 5 is ide ntical to lane 4 , but has been i ncub ated with 200 m M sodium acetate prior sample preparation. 374 T. Selmer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 previously shown that glutamate thiol ester-containing proteins, such as CoA-transferases and a2-macroglobulin, are site-speci®cally cleaved at elevated temperature [22]; a nucleophilic attack of the p eptidyl amide nitrogen of the glutamyl residue to the thiol ester carbonyl, releases the thiol forming a protein-bound 4-oxoproline residue. The peptide bond between this residue and the preceding amino acid is easily hydrolysed to yield a truncated pr otein and a C-terminal polypeptide, which is N-terminally blocked b y a pyroglutamyl residue [23]. To our knowledge, the propio- nate CoA-transferase is the ®rst CoA-transferase that has been partially puri®ed in this catalytic intermediate form. The protein exhibited rather broad signals for the single and double protonated m olecular ions in MALDI-TOF- MS measurements, indicating a molecular mass of 56 607  6 0 D a. The molecular mass of the transferase increased by 750 Da when the enzyme was incubated i n the presence of 100 l M propionyl-CoA prior to the measure- ments, indicating the f ormation of an enzyme CoA-thiol ester as a covalent catalytic intermediate. Cloning and sequencing of the gene encoding propionate CoA-transferase The enzyme w as blotted onto a poly(vinylidene di¯uoride) membrane and subjected to Edman degradation. Internal peptides were generated by cleavage with trypsin and puri®ed to homogeneity by RP-HPLC. Two of these peptides were sequenced by Edman degradation. A degen- erated primer pair was d educed from the N-terminus and from one of these peptides (Table 2), which was used to amplify a 3 00-bp fragment o f genomic DNA from C. pr o- pionicum by PCR. The fragment was cloned into a TOPO TA vector and sequenced. The sequence w as in accordance with the amino-acid sequences used for primer deduction and was similar to other CoA-transferases in the databases. A labelled PCR-product was used to screen a library of genomic DNA from C. propionicum in a k-ZAP-Express vector. Two clones were isolated and excised from the phage to yield the corresponding plasmid, which was subsequently sequenced. The clones contained identical inserts of 2.7-kb and e ncoded t he complete 524 amino-acid ORF corre- sponding to the propionate CoA-transferase (Fig. 4). According to the amino-acid sequence, an average molec- ular mass of 56 553 Da and a n isoelectric point of 4.9 1 was predicted for the encoded p rotein. B oth values w ere in agreement with the observed molecular mass and the elution of the enzyme during the ®nal puri®cation step. In addition to the CoA-transferase gene, a second ORF (lcdB), encoding 122 C-terminal amino acids of a protein similar to the 2-hydroxyglutaryl-CoA dehydratase b subunit of A. fermentans, was detected upstream of the transferase gene. This gene probably encodes one subunit of the (R)- lactoyl-CoA d ehydratase required in the reductive pathway from alanine to propionate. Directly downstream of the propionate CoA-transferase gene (pct), an AT-rich region was found that resembles rho-independent termination signals. The nucleotide sequence of the full insert has b een deposited under t he accession number AJ276553 in the EMBL nucleotide sequence database. Sequence analysis The amino-acid sequence of propionate CoA-transferase was compared to o ther proteins in the database using the BLAST algorithm [24,25]. The protein was most similar t o a putative acetoacetate:acetyl-CoA CoA-transferase from B. ha lodu rans (B84137, 56% identity, 519 amino-acid overlap [26]), and hypothetical proteins f rom E. coli (E85777 ydiF, 45% i dentity, 519 amino-acid overlap [ 27]), Aeropyrum pernix (D72478, 38% ident ity, 541 amino-acid overlap [28]), and Staphylococcus aureus (F89786, 36% identity, 519 amino-acid overlap [29]). Other hits were CoA-transferases from various microorganisms including Table 1. Puri®cation of pro pionate CoA-transferase from C. propionicum. The enzyme Activity was measured as described e arlier [6]. O ne unit of propionate CoA-transferase act ivity corresponds to the f ormation of 1 lmol a cetyl-CoA p er min from propionyl-CoA (100 l M ) a nd ace tate (200 m M )at25°C. No te that the activity in the cell free extract is the sum of CoA-transferase and phosphotransacetylase and that the l atter enzyme is completely s eparated from the CoA-transferase by t he ®rst colu mn. Puri®cation step Total activity (U) Protein (mg) Speci®c activity (Uámg )1 ) Puri®cation factor (fold) Yield (%) Cell free extract 3200 1391 2.3 1 100 Q-Sepharose 1560 46 34 15 49 Resource-Phe 1428 20 70 30 45 Resource-Q 1306 15 85 37 41 Table 2. N-terminal sequencing and PCR-primer deduction. The N-terminal amino acid sequences of the puri®ed, blotted protein and of two internal peptides are shown. These sequ ences have been used to deduce a degenerated prime r pair for ampli®cation of a propionate CoA-transferase speci®c probe f rom C. propionicum genomic DNA. M  AorC,N  A, G, C or T, R  AorG,H  A, C o r T, Y  CorT. Region/peptide Amino-acid sequence Deduced PCR primer N-Terminus MRKVPIITADEAAKLIK-D Sense: 5¢-ATGMGNAARGTNCCNATHATHACN GCNGAYGCTGC-3¢ Peptide 1 YIAGHWATVPALGK Antisense: 5¢)CCNARNGCNGGNCANATNGCC-3¢ Peptide 2 GTYADESGNITFEKEVAPLEGTSV-QA Not used Ó FEBS 2002 Propionate CoA-transferase (Eur. J. Biochem. 269) 375 Deinococcus radiodurans, B acillus s ubtilis, Streptomyces coelicolor, Heliobacter pylori, Mycobacterium tuberculo- sis, Haemophilus in¯uenzae and Clostridium acetobutylicum. These latter e nzymes belong to the 3 -oxoadipate CoA- transferase protein superfam ily and consist of t wo diff erent subunits. The similarity of these latter sequences to propi- onate CoA-tansferase was lower (23±28%) and restricted to the larger subunit of these enzymes (232±255 amino-acids overlap). However, when the C-terminal half of the amino- acid sequence of propionate-CoA-transferase was used for database search, t his part o f the sequ ence showed similarity to the smaller subunits of the latter enzymes. The catalytic glutamate residue of hetero-oligomeric enzymes b elonging to the 3-oxoadipate CoA-transferase superfamily is found in the small subun it and is located within a so-called (S)ENG motif [11]. This characteristic motif is not found in the sequence of propionate CoA- transferase or i n any of the putative proteins from B. halodurans, E. coli, A. pe rnix or S. aureus.Therefore,a multiple sequence alignment o f the b subunits of 3-oxoadi- pate CoA-transferase a nd the C -terminal h alf (starting with Leu276) of propionate CoA-transferase from C. propioni- cum was generated using CLUSTALW . The most likely candidate for the catalytic glutamate of propionate CoA- transferase based on these data was glutamate 324 (Fig. 3 ). Detection of glutamate 324 as the catalytic carboxylate of propionate CoA-transferase As the sequence analysis d id not allow an unequivocal identi®cation of the catalytic glutamate of propionate CoA- transferase, this residue was s peci®cally labelled. The thiol ester in the proposed enzyme-CoA intermediate of the CoA- transferase reaction cycle is more reactive than, for example, free propionyl-CoA. This higher reactivity allows the reduction of the thiol ester with sodium borohydride to yield a protein-bound 2-amino-5-hydroxyvaleryl r esidue [11]. In addition, it has been shown that nucleophiles such as methylamine or hydroxylamine can cleave enzyme-bound thiol esters [22], yielding N-methylglutamine or the corre- sponding hyd roxamic acid. T hese reactions are useful tools for identifying the c atalytic residue, a s the derivat ives give rise to changes in the molecular m asses o f peptides that originate from the protein inactivated by either borohydride ()14 Da) or hydroxylamine (+15 Da). When propionate CoA-transferase was incubated with 100 l M propionyl-CoA in the presence of either sodium borohydride ( 20 m M ) o r hydroxylamine (pH 7.5, 2 00 m M ), the en zyme w as rapidly and irreversibly inactivated. The inactivation was strictly dependent on the presence of propionyl-CoA. The inactivated proteins and controls, which had been incubated with the reagents but without propionyl-CoA, were subjected to reductive carboxymethylation and desalt- ed by gel ®ltration. Aliquots of t hese samples were digested for 16 h at 37 °C with 2% (w/w) of either chymotrypsin, endoprotease-AspN, en doprotease-GluC o r trypsin. The peptides were analysed by MALDI-TOF-MS without puri®cation. Altho ugh only around 30±50% of the predict- ed peptides were detected in one particular digest, all four samples together c overed the full amino-acid sequence predicted by the gene. The molecular masses of peptides carrying the proposed catalytic glutamate 324 were found in all s amples except t he endoprotease-GluC digest. The masses of these peptides exclusively s howed differences for inactivate d samples and controls. As summarized in T able 3, the derivatives showed the predicted mass differences of )14 Da and +15 Da for sodium borohydri de and hydroxylamine inactivated enzyme, r espectively. In particular the observation of a chymotryptic peptide comprising amino acids 322±338 was very crucial, since this peptide contains the glutamate 324 as the sole c arboxylate. Therefore, the tentative assignment of glutamate 324 has been con®rmed by these experiments. DISCUSSION Propionate CoA-transferase from C. propionicum has been puri®ed and initially characterized previously [6]. In this communication we report an i mproved puri®cation proto- col for the enzyme. The gene encoding the protein was cloned, sequenced and g lutamate 324 was identi®ed as the active site glutamate residue. The gene encoding propionate CoA-transferase from C. propionicum was cloned and sequenced. The encoded protein was similar to CoA-transferases belonging to the Fig. 3. Sequence alignment of t he active site region of various h eterooligomeric CoA-transferases and propionate CoA-transferase. The amino acid sequences surrounding the characteristic (S)ENG-motif of the former proteins is shown. Note that this motif is not easily recognized in propionate CoA-transferase from C. propionicum (Cpro). The consensus sequence shows amino acids which are conserved in at least eight out of 12 enzymes. Pput, Pseudomo nas putida; Acal, Acinetobacter calcoaceticus; Drad, Deinococcus radiodurans; Bsub, Bacillus subtilis; Scoe, Streptomyces coelicolor; Hpyl, Heliobacter pylori; Mtub, Mycobacterium tuberculosis; Hinf, Haemophilus in¯uenzae; Cace, Clostridium acetobutulicum. 376 T. Selmer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 3-oxoadipate CoA-transferase s uperfamily. However, whereas these proteins consist of two subunits and contain a highly indicative ®ngerprint motif [(S)ENG] surrounding the a ctive centr e glutamate, p ropionate CoA- transferase consists of one polypep tide and the ®ngerprint m otif is not found in this protein. A site-speci®c label of the catalytic glutamate via the thiol ester catalytic intermediate , either by reductive cleavage with borohydride or by cleavage with hydroxylamine allowed the identi®cation o f the active site carboxylate. The predicted derivatives were located exclu- sively on glutamate 324, which led us to conclude that this residue is the active site carboxylate. The proteins most similar to propionate CoA-transfer- ase in the databases are a putative acetoacetate CoA- transferase from Bacillus halodurans and o ther proteins with as yet u nknown function from Escherichia coli, Aeropyrum pernix and Staphylococcus aureus. O ur data strongly suggest that these genes encode CoA-transferases. As shown in F ig. 4 , the proteins align well, and in particular the glutamate residue 324 is c onserved a mong all these proteins. It seems therefore reasonable to conclude that these proteins form a novel subclass of CoA-transferases. These en zymes lack t he characteristic (S)ENG consensus motif of members of th e 3-oxoadipate CoA-transferase superfamily and e xhibit either a homool- igomeric or monomeric quarternary structure. It is reasonable to s uggest that a gene fusion could h ave occurred during the evolution of the former enzymes. Such a natural g ene fusion has also been suggested for the mammalian oxoadipate CoA-transferase [30]. In agree- ment with this proposal, i t has been shown t hat the two subunits of glutaconate CoA-transferase from A. fermen- tans could b e f used with genetic tools to y ield an active enzyme composed of a single polypeptide [31]. Fig. 4. Sequence alignment of propionate CoA-transferase and similar p roteins derived from genome projects. The p ropionate CoA- transferase from Clostridium propionicum (Cpro) is compared to proteins encoded in the genomes o f Bacillus h alodurans (Bhal), Eschrichia coli (Ecol) and Aeropyrum pernix (Aper). The sequ ence s have b een align ed using CLUSTALW . Residues i dentical in at least three proteins are s haded. Invariant p ositions are marked with asterisks a nd conservative exchanges a re marked by dots. The catalytic glutamate is s hown in bold and a mino acid con®rmed b y amino a cid sequencing for propionate CoA-transferase are underlined. Ó FEBS 2002 Propionate CoA-transferase (Eur. J. Biochem. 269) 377 Despite the low sequence similarities of different CoA- transferases on the amino acid sequence level, CoA- transferases have been predicted to h ave a very similar f old [17]. In agreement with this proposal, we found that the secondary structural elements predicted f or the amino-acid sequence of propionate CoA-transferase superimpose very well with the known elements in the crystal structure of glutaconate CoA-transferase ( Fig. 5). Nevertheless, there are some s triking differences in the arrangement of t he secondary structural elements, which can partially be explained by the known biochemical properties of the enzymes. As shown in Fig. 5 A,B, the secondary structure elements in glutaconate CoA-transferase form two ou ter layers of a helices followed by one layer of b sheets a nd an inner l ayer of ahelices. This arrangement is found in bo th subunits of the protein. I t is r emarkable that the outer layer of a helices in the large subunit of glutaconate CoA- transferase (Fig. 5A) is apparently missing in pr opionate CoA-transferase ( Fig. 5C). The crystal str ucture has shown that two antiparallel b sheets (Fig. 5B, triangles 6 and 7, respectively) of the b subunit of g lutaconate CoA-tr ansfer- ase p rotrude into a cleft on the s urface of the a subunit a nd are involved in mediation of subunit interactions. Therefore, the lack of this e lement (Fig. 5C) is not surprising when the homooligomeric structure o f propionate CoA-transferase is taken i nto a ccount. A n a dditional i nteresting difference between both structures i s the lack of a subdomain formed by two b sheets and one a helix connecting the bsheet 2 of the b subunit, which carries the catalytic glutamate, with the inner l ayer of a helices (Fig. 5B, triangles 3 and 4 and circle 3, respectively). This subdomain is thought to be involved in the substrate binding of glutaconate CoA-transferase. It has been suggested that two speci®c serine residues, Ser78 (in subunit A) and Ser68 (in subunit B ), are involved i n the formation of hydrogen bonds with the e-carboxylate of glutaryl-CoA and t hat the latter s erine is located within this subdomain. It is remarkable that both residues are appar- ently replaced by stretches of rather hydrophobic residues in propionate CoA-transferase and it is reasonable to conclude that the additional subdomain in glutaconate CoA-trans- Table 3. Mass spectrometry of peptides derived from controls and i nactivated p ropionate CoA -transferase. Propionate CoA-transferase w as i ncu- bated in the absence (control) or presence of propionyl-CoA with either hydroxylamine (200 m M ) or sodium borohydride (20 m M ) as described in Material and methods. The prote in was reductivel y carboxymethylated and subsequently digested with the proteases as indicated. The p eptides were analysed by MALDI-TOF-MS as described elsewhere [14]. The molecular masses expected for the controls (E 324  COOH), hydroxyl- amine- (E 324  CONHOH) or borohydride-treated (E 324  CH 2 OH) peptides is given in parentheses. The catalytic glutamate residue is shown in bold. Unless stated otherwise, the measured and calculated molecular masses are given as monoisotopic masses. For trypsin, both calculated and measured va lues refer t o the average mass. Digest Sequence Control Hydroxylamine (+15 Da) Borohydride ()14 Da) Chymotrypsin 322 TAESGAIGGVPAGGVRF 338 1547 (1547) Da 1563 (1562) Da 1533 (1533) Da Endoproteinase-AspN 317 DFMTLTAESGAIGGVPAGGVRFGASYNA 344 2718 (2717) Da 2733 (2732) Da 2705 (2703) Da Trypsin 286 GAIELEKDVAVNLGVGAPEYVASVADEEG IVDFMTLTAESGAIGGVPAGGVR 337 5107 (5105) Da 5122 (5120) Da 5094 (5091) Da Fig. 5. Structural similarity of glutaconate CoA-transferase (A,B) [15] and the predicted topology of secondary structure elements in propionate CoA- transferase (C). The topological diagram shows the arrangement of secondary structure elements. These structure elements have been predicted for propionate CoA-transferase by the program 3 D - PSSM provided by the Imperial Cancer Research Fold Recognition Server. These elements have been aligned to the structure of glutaconate CoA-transferase. Elements homologous to the a subunit of glutaconate CoA-transferase are given in black, those h omologous to the b subunit in white. T he a helices are shown by circles a nd the bsh eets by triangles. The position of t he catalytic glutamates is marked by a dot. 378 T. Selmer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ferase represents an adaptation for t he binding o f a dicarboxyl-CoA by the latter enzyme. During the course of our research, an interesting obser- vation was made. A ll attempts to exp ress the cloned gene from C. propionicum in E. coli failed (A. Willanzheimer, unpublished observations) 22 ; the transformed E. coli cells carrying an isopropyl thio-b- D -galactoside-inducible expres- sion vector exhibited no g rowth defect until the p rotein was induced by isopropyl thio-b- D -galactoside. Upon induction of the protein, E. coli stopped growing. T hese observations may point to a severe impairment of the cellular metabolism of E. coli by the C. prop ionicum enzyme. 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Propionate CoA-transferase from Clostridium propionicum Cloning of the gene and identi®cation of glutamate 324 at the active site Thorsten. for the catalytic glutamate of propionate CoA- transferase based on these data was glutamate 324 (Fig. 3 ). Detection of glutamate 324 as the catalytic carboxylate