Crystal Structure and Molecular Mechanism of Phosphotransbutyrylase from Clostridium acetobutylicum.

Acetone-butanol-ethanol (ABE) fermentation by the anaerobic bacterium Clostridium acetobutylicum has been considered a promising process of industrial biofuel production. Phosphotransbutyrylase (phosphate butyryltransferase, PTB) plays a crucial role in butyrate metabolism by catalyzing the reversible conversion of butyryl-CoA into butyryl phosphate. Here, we report the crystal structure of PTB from the Clostridial host for ABE fermentation, C. acetobutylicum, (CaPTB) at a 2.9 Å resolution. The overall structure of the CaPTB monomer is quite similar to those of other acyltransferases, with some regional structural differences. The monomeric structure of CaPTB consists of two distinct domains, the N- and C-terminal domains. The active site cleft was formed at the interface between the two domains. Interestingly, the crystal structure of CaPTB contained eight molecules per asymmetric unit, forming an octamer, and the size-exclusion chromatography experiment also suggested that the enzyme exists as an octamer in solution. The structural analysis of CaPTB identifies the substrate binding mode of the enzyme and comparisons with other acyltransferase structures lead us to speculate that the enzyme undergoes a conformational change upon binding of its substrate.

Two propanediol utilization-like proteins of Moorella thermoacetica with phosphotransacetylase activity.

Moorella thermoacetica is one of the model acetogenic bacteria for the resolution of the Wood-Ljungdahl (acetyl-CoA) pathway in which CO2 is autotrophically assimilated yielding acetyl-CoA as central intermediate. Its further conversion into acetate relies on subsequent phosphotransacetylase (PTA) and acetate kinase reactions. However, the genome of M. thermoacetica contains no pta homologous gene. It has been speculated that the moth_0864 and moth_1181 gene products sharing similarities with an evolutionarily distinct phosphotransacylase involved in 1,2-propanediol utilization (PDUL) of Salmonella enterica act as PTAs in M. thermoacetica. Here, we demonstrate specific PTA activities with acetyl-CoA as substrate of 9.05 and 2.03 U/mg for the recombinant enzymes PDUL1 (Moth_1181) and PDUL2 (Moth_0864), respectively. Both showed maximal activity at 65 °C and pH 7.6. Native proteins (90 kDa) are homotetramers composed of four subunits with apparent molecular masses of about 23 kDa. Thus, one or both PDULs of M. thermoacetica might act as PTAs in vivo catalyzing the penultimate step of the Wood-Ljungdahl pathway toward the formation of acetate. In silico analysis underlined that up to now beside of M. thermoacetica, only Sporomusa ovata contains only PDUL like class(III)-PTAs but no other phosphotransacetylases or phosphotransbutyrylases (PTBs).

Potential Role of Acetyl-CoA Synthetase (acs) and Malate Dehydrogenase (mae) in the Evolution of the Acetate Switch in Bacteria and Archaea.

Although many Archaea have AMP-Acs (acetyl-coenzyme A synthetase) and ADP-Acs, the extant methanogenic genus Methanosarcina is the only identified Archaeal genus that can utilize acetate via acetate kinase (Ack) and phosphotransacetylase (Pta). Despite the importance of ack as the potential urkinase in the ASKHA phosphotransferase superfamily, an origin hypothesis does not exist for the acetate kinase in Bacteria, Archaea, or Eukarya. Here we demonstrate that Archaeal AMP-Acs and ADP-Acs contain paralogous ATPase motifs previously identified in Ack, which demonstrate a novel relation between these proteins in Archaea. The identification of ATPase motif conservation and resulting structural features in AMP- and ADP-acetyl-CoA synthetase proteins in this study expand the ASKHA superfamily to include acetyl-CoA synthetase. Additional phylogenetic analysis showed that Pta and MaeB sequences had a common ancestor, and that the Pta lineage within the halophilc archaea was an ancestral lineage. These results suggested that divergence of a duplicated maeB within an ancient halophilic, archaeal lineage formed a putative pta ancestor. These results provide a potential scenario for the establishment of the Ack/Pta pathway and provide novel insight into the evolution of acetate metabolism for all three domains of life.

In silico study and validation of phosphotransacetylase (PTA) as a putative drug target for Staphylococcus aureus by homology-based modelling and virtual screening.

Staphylococcus aureus, a Gram-positive bacterium, can cause a range of illnesses from minor skin infections to life-threatening diseases, such as bacteraemia, endocarditis, meningitis, osteomyelitis, pneumonia, toxic shock syndrome and sepsis. Due to the emergence of antibiotic resistance strains, there is a need to develop of new class of antibiotics or drug for this pathogen. The phosphotransacetylase enzyme plays an important role in the acetate metabolism and found to be essential for the survival of the S. aureus. This enzyme was evaluated as a putative drug target for S. aureus by in silico analysis. The 3D structure of the phosphotransacetylase from S. aureus was modelled, using the 1TD9 chain 'A' from Bacillus subtilis as a template at the resolution of 2.75 Å. The generated model has been validated by PROCHECK, WHAT IF and SuperPose. The docking was performed by the Molegro virtual docker using the ZINC database generated ligand library. The ligand library was generated within the limitation of the Lipinski rule of five. Based on the dock-score, five molecules have been subjected to ADME/TOX analysis and subjected for pharmacophore model generation. The zinc IDs of the potential inhibitors are ZINC08442078, ZINC8442200, ZINC 8442087 and ZINC 8442184 and found to be pharmacologically active antagonist of phosphotransacetylase. The molecules were evaluated as no-carcinogenic and persistent molecule by START programme.

Cloning, characterization and transcriptional analysis of two phosphate acetyltransferase isoforms from Azotobacter vinelandii.

Acetate is abundant in soil contributing to a great extent on carbon cycling in nature. Phosphate acetyltransferase (Pta, EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl-P to CoA forming acetyl-CoA and inorganic phosphate, participating to acetate assimilation/dissimilation reactions. In the present study, we demonstrate that Azotobacter vinelandii, a nitrogen-fixing, free-living, soil bacterium, possesses two class II phosphate acetyltransferase isoforms, AvPTA-1 and AvPTA-2, with different kinetic properties. At the acetyl-CoA forming direction, AvPTA-1 has lower affinity for acetyl-P and higher affinity for CoA than AvPTA-2 while at the acetyl-P forming direction; activity was measured only for AvPTA-1. Quantification of their expression patterns by RT-qPCR indicated that both genes are expressed during exponential growth on glucose or acetate and are down-regulated in the stationary phase. The ammonium availability during acetate growth resulted in up-regulation of Avpta-2 expression only. Further, the gene expression patterns of other related gene transcripts were also investigated, in order to understand the influence of each pathway in the assimilation/dissimilation of acetate.

Preliminary structural studies on the MtxX protein from Methanococcus jannaschii.

Methanococcus jannaschii has an mtr gene cluster expressing N(5)-methyltetrahydromethanopterin:coenzyme M methyltransferase, which generates methane by reducing CO(2) with H(2) with concomitant energy production under strictly anaerobic conditions. Some methanogenic archaea also have an mtr gene-cluster homologue, the mtxXAH gene cluster. M. jannaschii has both an entire mtr gene cluster and a single mtxX gene instead of the whole mtxXAH gene cluster. A PSI-BLAST search, secondary-structure prediction and the absence of phosphotransacetylase activity in M. jannaschii strongly support the possibility that the MtxX protein constitutes a unique methyltransferase family. In this study, the MtxX protein from M. jannaschii has been cloned, expressed, purified and crystallized. Synchrotron data were collected to 2.9 A from a crystal of selenomethionine-substituted MtxX protein. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 54.9, b = 54.9, c = 341.1 A, beta = 120.0 degrees . A full structure determination is under way in order to provide insight into the structure-function relationship of this protein.

Analyses of the acetate-producing pathways in Corynebacterium glutamicum under oxygen-deprived conditions.

Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetyl-coenzyme A (CoA). Simultaneous disruption of pta, encoding phosphotransacetylase, and ack, encoding acetate kinase, resulted in no measurable change in acetate productivity. A mutant strain with disruptions in pta, ack and as-yet uncharacterized gene (cgR2472) exhibited 65% reduced acetate productivity compared to the parental strain, although a single disruption of cgR2472 exhibited no effect on acetate productivity. The gene cgR2472 was shown to encode a CoA-transferase (CTF) that catalyzes the formation of acetate from acetyl-CoA. These results indicate that PTA-ACK as well as CTF is involved in acetate production in C. glutamicum. This study provided basic information to reduce acetate production under oxygen-deprived conditions.

In vivo and in vitro analyses of single-amino acid variants of the Salmonella enterica phosphotransacetylase enzyme provide insights into the function of its N-terminal domain.

The function of the N-terminal domain ( approximately 350 residues) of the Pta (phosphotransacetylase) enzyme of Salmonella enterica is unclear. Results from in vivo genetic and in vitro studies suggest that the N-terminal domain of Pta is a sensor for NADH and pyruvate. We isolated 10 single-amino acid variants of Pta that, unlike the wild-type protein, supported growth of a strain of S. enterica devoid of Acs (acetyl-CoA synthetase; AMP-forming) activity on 10 mm acetate. All mutations were mapped within the N-terminal domain of the protein. Kinetic analyses of the wild type and three variant Pta proteins showed that two of the variant proteins were faster enzymes (k(cat) 2.5-3-fold > k(cat) Pta(WT). Results from sedimentation equilibrium experiments are consistent with Pta(WT) being a trimer. Pta variants formed more hexamer than the Pta(WT) protein. NADH inhibited Pta(WT) activity by inducing a conformational change detectable by limited trypsin proteolysis; NADH did not inhibit variant protein Pta(R252H). Pyruvate stimulated Pta(WT) activity, and its effect was potentiated in the variants, being most pronounced on Pta(R252H).

Structural and functional studies suggest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila.

Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. Two crystal structures of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila in complex with the substrate CoA revealed one CoA (CoA1) bound in the proposed active site cleft and an additional CoA (CoA2) bound at the periphery of the cleft. The results of isothermal titration calorimetry experiments are described, and they support the hypothesis that there are distinct high-affinity (equilibrium dissociation constant [KD], 20 microM) and low-affinity (KD, 2 mM) CoA binding sites. The crystal structures indicated that binding of CoA1 is mediated by a series of hydrogen bonds and extensive van der Waals interactions with the enzyme and that there are fewer of these interactions between CoA2 and the enzyme. Different conformations of the protein observed in the crystal structures suggest that domain movements which alter the geometry of the active site cleft may contribute to catalysis. Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for Ser309 and Arg310, which are proximal to the reactive sulfhydryl of CoA1. The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue Asp316, followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate. We propose that Arg310 binds acetyl phosphate and orients it for optimal nucleophilic attack. The hypothesized mechanism proceeds through a negatively charged transition state stabilized by hydrogen bond donation from Ser309.

Crystal structures of a phosphotransacetylase from Bacillus subtilis and its complex with acetyl phosphate.

Phosphotransacetylase (Pta) [EC 2.3.1.8] plays a major role in acetate metabolism by catalyzing the reversible transfer of the acetyl group between coenzyme A (CoA) and orthophosphate: CH(3)COSCoA+HPO(4)(2-)<-->CH(3)COOPO(3)(2-) +CoASH. In this study, we report the crystal structures of Pta from Bacillus subtilis at 2.75 A resolution and its complex with acetyl phosphate, one of its substrates, at 2.85 A resolution. In addition, the Pta activity of the enzyme has been assayed. The enzyme folds into an alpha/beta architecture with two domains separated by a prominent cleft, very similar to two other known Pta structures. The enzyme-acetyl phosphate complex structure reveals a few potential substrate binding sites. Two of them are located in the middle of the interdomain cleft: each one is surrounded by a region of strictly and highly conserved residues. High structural similarities are found with 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA), and isocitrate and isopropylmalate dehydrogenases, all of which utilize NADP+ as their cofactor, which binds in the interdomain cleft. Their substrate binding sites are close to the acetyl phosphate binding sites of Pta in the cleft as well. These results suggest that the CoA is likely to bind to the interdomain cleft of Pta in a similar way as NADP+ binds to the other three enzymes.

Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation.

Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A nonreplicative integrational plasmid containing the phosphotransacetylase gene (pta) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette were constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the pta-deleted mutant (PTA-Em), which was confirmed by Southern hybridization. SDS-PAGE and two-dimensional protein electrophoresis results indicated that protein expression was changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and acetate kinase (AK) in the mutant were reduced by more than 60% for PTA and 80% for AK. The mutant grew more slowly in batch fermentation with glucose as the substrate but produced 15% more butyrate and 14% less acetate as compared to the wild-type strain. Its butyrate productivity was approximately 2-fold higher than the wild-type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition, and the final butyrate concentration was improved by 68%. However, inactivation of pta gene did not completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first-reported genetic engineering study demonstrating the feasibility of using a gene-inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum in order to improve butyric acid production from glucose.

Acidic proteome of growing and resting Lactococcus lactis metabolizing maltose.

The acidic proteome of Lactococcus lactis grown anaerobically was compared for three different growth conditions: cells growing on maltose, resting cells metabolizing maltose, and cells growing on glucose. In maltose metabolizing cells several proteins were up-regulated compared with glucose metabolizing cells, however only some of the up-regulated proteins had apparent relation to maltose metabolism. Cells growing on maltose produced formate, acetate and ethanol in addition to lactate, whereas resting cells metabolizing maltose and cells growing on glucose produced only lactate. Increased levels of alcohol-acetaldehyde dehydrogenase (ADH) and phosphate acetyltransferase (PTA) in maltose-growing cells compared with glucose-growing cells coincided with formation of mixed acids in maltose-growing cells. The resting cells did not grow due to lack of an amino acid source and fermented maltose with lactate as the sole product, although ADH and PTA were present at high levels. The maltose consumption rate was approximately three times lower in resting cells than in exponentially growing cells. However, the enzyme levels in resting and growing cells metabolizing maltose were similar, which indicates that the difference in product formation in this case is due to regulation at the enzyme level. The levels of 30S ribosomal proteins S1 and S2 increased with increasing growth rate for resting cells metabolizing maltose, maltose-growing cells and glucose-growing cells. A modified form of HPr was synthesized under amino acid starvation. This is suggested to be due to alanine misincorporation for valine, which L. lactis is auxotrophic for. L. lactis conserves the protein profile to a high extent, even after prolonged amino acid starvation, so that the protein expression profile of the bacterium remains almost invariant.

Crystal structure of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila.

Phosphotransacetylase (Pta) [EC 2.3.1.8] is ubiquitous in the carbon assimilation and energy-yielding pathways in anaerobic prokaryotes where it catalyzes the reversible transfer of the acetyl group from acetyl phosphate to CoA forming acetyl CoA and inorganic phosphate. The crystal structure of Pta from the methane-producing archaeon Methanosarcina thermophila, representing the first crystal structure of any Pta, was determined by multiwavelength anomalous diffraction at 2.7 A resolution. In solution and in the crystal, the enzyme forms a homodimer. Each monomer consists of two alpha/beta domains with a cleft along the domain boundary, which presumably contains the substrate binding sites. Comparison of the four monomers present in the asymmetric unit indicates substantial variations in the relative orientation of the two domains and the structure of the putative active site cleft. A search for structural homologs revealed the NADP(+)-dependent isocitrate and isopropylmalate dehydrogenases as the only homologs with a similar two-domain architecture.

Expression, purification, crystallization and preliminary X-ray analysis of phosphotransacetylase from Methanosarcina thermophila.

Phosphotransacetylase (Pta) from the anaerobic archaeon Methanosarcina thermophila has been heterologously expressed in a soluble form which facilitated crystallization using the hanging-drop vapor-diffusion method with ammonium sulfate as a precipitant. This is the first report of the crystallization of any Pta. While the M. thermophila Pta has high sequence identity to Ptas from other organisms, it has no homology to any previously crystallized proteins. The protein crystallized in space group I4(1), with unit-cell parameters a = b = 114.8, c = 127.8 A, alpha = beta = gamma = 90 degrees. The crystals diffracted to 2.5 A resolution using Cu Kalpha radiation. The enzyme had previously been reported to exist as a monomer; however, the self-rotation function showed the presence of a non-crystallographic symmetry axis at psi = 90, phi = 90, kappa = 180 degrees, suggesting oligomerization. Dynamic light-scattering analysis supported a dimeric state for Pta in solution.

Cloning of the phosphotransacetylase gene from Lactobacillus sanfranciscensis and characterization of its gene product.

The phosphotransacetylase (PTA) (EC 2.3.1.8) catalyzes a key branch point reaction in the carbohydrate pathway of Lactobacillus sanfranciscensis. In this report, we describe the cloning of the pta gene. The DNA sequence analysis revealed a 987 bp open reading frame encoding a protein with a molecular mass of 35.5 kD. These are the first studies on a PTA of an organism representative for the heterofermentative lactic acid bacteria. Unlike in most other bacteria analysed so far, in L. sanfranciscensis the pta gene is not adjacent located to the gene encoding acetate-kinase. The PTA was heterologously expressed as a biotinylated fusion protein in E. coli and purified to homogeneity. Rate dependence on all substrates followed Michaelis-Menten kinetics. The apparent Km values for acetylphosphate and CoA (forward reaction) were 1.3 and 0.1 mM, respectively. The apparent Vmax was 194 U/mg. The enzyme also catalyzed in vitro the reverse reaction with apparent Km values for acetylCoA and phosphate of 0.6 and 6.7 mM, respectively (Vmax of 38 U/mg). The PTA showed a wide range of temperature for optimal activity (49 degrees C to 58 degrees C). It was inactivated after 15 min at 60 degrees C. Its activity was not affected by addition of MgCl2 (10 mM) or KCl (100 mM).

The synthetase domains of cobalamin biosynthesis amidotransferases cobB and cobQ belong to a new family of ATP-dependent amidoligases, related to dethiobiotin synthetase.

Phosphotransacetylases of Escherichia coli and several other bacteria contain an additional 350-aa N-terminal fragment that is not required for phosphotransacetylase activity. Sequence analysis of this fragment revealed that it is closely related to a family of ATP-dependent enzymes that also includes dethiobiotin synthetase and the synthetase domains of two amidotransferases involved in cobalamin biosynthesis, cobyrinic acid a,c-diamide synthase (CobB) and cobyric acid synthase (CobQ). Further database searches showed that this enzyme family is also related to the MinD family of ATPases involved in regulation of cell division in bacteria and archaea. Analysis of sequence conservation in the members of this enzyme family using the structure of dethiobiotin synthetase active site as a guide allowed us to suggest a model for the interaction of CobB and CobQ with their respective substrates. CobB and CobQ were also found to contain unusual Triad family (class I) glutamine amidotransferase domains with conserved Cys and His residues, but lacking the Glu residue of the catalytic triad. These results should help in understanding the enzymology of cobalamin biosynthesis and in resolving the role of phosphotransacetylase in regulation of the carbon flow to and from acetate.

Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase and acetate kinase.

The Corynebacterium glutamicum ack and pta genes encoding the acetate-activating enzymes acetate kinase and phosphotransacetylase were isolated, subcloned on a plasmid and re-introduced into Corynebacterium glutamicum. Relative to the wild-type, the recombinant strains showed about tenfold higher specific activities of both enzymes. Sequence analysis of a 3657 bp DNA fragment revealed that the ack and pta genes are contiguous in the corynebacterial chromosome, with pta upstream and the last nucleotide of the pta stop codon (TAA) overlapping the first of the ack start codon (ATG). The predicted gene product of pta consists of 329 amino acids (Mr 35242), that of ack consists of 397 amino acids (Mr 43098) and the amino acid sequences of the two polypeptides show up to 60 % (phosphotransacetylase) and 53% (acetate kinase) identity in comparison with respective enzymes from other organisms. Northern (RNA) blot hybridizations using pta- and ack-specific probes and transcriptional cat fusion experiments revealed that the two genes are transcribed as a 2.5 kb bicistronic mRNA and that the expression of this operon is induced when Corynebacterium glutamicum grows on acetate instead of glucose as a carbon source. Directed inactivation of the chromosomal pta and ack genes led to the absence of detectable phosphotransacetylase and acetate kinase activity in the respective mutants and to their inability to grow on acetate. These data indicate that no isoenzymes of acetate kinase and phosphotransacetylase are present in Corynebacterium glutamicum and that a functional acetate kinase/phosphotransacetylase pathway is essential for growth of this organism on acetate.

Sequence and organization of genes encoding enzymes involved in pyruvate metabolism in Mycoplasma capricolum.

The region of the genome of Mycoplasma capricolum upstream of the portion encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced. Examination of the sequence revealed open reading frames corresponding to numerous genes involved with the oxidation of pyruvate. The deduced gene organization is naox (encoding NADH oxidase)-lplA (encoding lipoate-protein ligase)-odpA (encoding pyruvate dehydrogenase EI alpha)-odpB (encoding pyruvate dehydrogenase EI beta)-odp2(encoding pyruvate dehydrogenase EII)-dldH (encoding dihydrolipoamide dehydrogenase)-pta (encoding phosphotransacetylase)-ack (encoding acetate kinase)-orfA (an unknown open reading frame)-kdtB-ptsI-crr. Analysis of the DNA sequence suggests that the naox and lplA genes are part of a single operon, odpA and odpB constitute an additional operon, odp2 and dldH a third operon, and pta and ack an additional transcription unit. Phylogenetic analyses of the protein products of the odpA and odpB genes indicate that they are most similar to the corresponding proteins from Mycoplasma genitalium, Acholeplasma laidlawii, and Gram-positive organisms. The product of the odp2 gene contains a single lipoyl domain, as is the case with the corresponding proteins from M. genitalium and numerous other organisms. An evolutionary tree places the M. capricolum odp2 gene product in close relationship to the corresponding proteins from A. laidlawii and M.genitalium. The dldH gene encodes an unusual form of dihydrolipoamide dehydrogenase that contains an aminoterminal extension corresponding to a lipoyl domain, a property shared by the corresponding proteins from Alcaligenes eutrophus and Clostridium magnum. Aside from that feature, the protein is related phylogenetically to the corresponding proteins from A. laidlawii and M. genitalium. The phosphotransacetylase from M. capricolum is related most closely to the corresponding protein from M. genitalium and is distinguished easily from the enzymes from Escherichia coli and Haemophilus influenzae by the absence of the characteristic amino-terminal extension. The acetate kinase from M. capricolum is related evolutionarily to the homologous enzyme from M. genitalium. Map position comparisons of genes encoding proteins involved with pyruvate metabolism show that, whereas all the genes are clustered in M. capricolum, they are scattered in M. genitalium.

Cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from Clostridium acetobutylicum ATCC 824.

The enzymes phosphotransacetylase (PTA) and acetate kinase (AK) catalyze the conversion of acetyl coenzyme A to acetate in the fermentation of Clostridium acetobutylicum. The acetate-producing step is an important element in the acidogenic fermentation stage and generates ATP for clostridial cell growth. The genes pta and ack, encoding PTA and AK, respectively, were cloned and sequenced. Enzyme activity assays were performed on cell extracts from Escherichia coli and C. acetobutylicum harboring the subclone, and both AK and PTA activities were shown to be elevated. DNA sequence analysis showed that the pta and ack genes are adjacent in the clostridial chromosome, with pta upstream. The pta gene encodes a protein of 333 amino acid residues with a calculated molecular mass of 36.2 kDa, and ack encodes a polypeptide of 401 residues with a molecular mass of 44.3 kDa. Primer extension analysis identified a single transcriptional start site located 70 bp upstream of the start codon for the pta gene, suggesting an operon arrangement for these tandem genes. The results from overexpression of ack and pta in C. acetobutylicum showed that the final ratios of acetate to other major products were higher and that there was a greater proportion of two- versus four-carbon-derived products.