Search

α-Methylacyl-CoA racemase in M. tuberculosis (MCR) has an essential role in fatty acid metabolism and cholesterol utilization, contributing to the bacterium's survival and persistence. Understanding the enzymatic activity and structural features of MCR provides insights into its physiological and pathological significance and potential as a therapeutic target. Here, we report high-resolution crystal structures for wild-type MCR in a new crystal form (at 1.65 Å resolution) and for three active-site mutants, H126A, D156A and E241A, at 2.45, 1.64 and 1.85 Å resolutions, respectively. Our analysis of the new wild-type structure revealed a similar dimeric arrangement of MCR molecules to that previously reported and details of the catalytic site. The determination of the structures of these H126A, D156A and E241A mutants, along with their detailed kinetic analysis, has now allowed for a rigorous assessment of their catalytic properties. No significant change outside the enzymatic active site was observed in the three mutants, establishing that the diminution of catalytic activity is mainly attributable to disruption of the catalytic apparatus involving key hydrogen bonding and water-mediated interactions. The wild-type structure, together with detailed mutational and biochemical data, provide a basis for understanding the catalytic properties of this enzyme, which is important for the design of future anti-tuberculosis drug molecules. [ABSTRACT FROM AUTHOR]

(R)-Benzylsuccinate is generated in anaerobic toluene degradation by the radical addition of toluene to fumarate and further degraded to benzoyl-CoA by a β-oxidation pathway. Using metabolic modules for benzoate transport and activation to benzoyl-CoA and the enzymes of benzylsuccinate β-oxidation, we established an artificial pathway for benzylsuccinate production in Escherichia coli, which is based on its degradation pathway running in reverse. Benzoate is supplied to the medium but needs to be converted to benzoyl-CoA by an uptake transporter and a benzoate-CoA ligase or CoA-transferase. In contrast, the second substrate succinate is endogenously produced from glucose under anaerobic conditions, and the constructed pathway includes a succinyl-CoA:benzylsuccinate CoA-transferase that activates it to the CoA-thioester. We present first evidence for the feasibility of this pathway and explore product yields under different growth conditions. Compared to aerobic cultures, the product yield increased more than 1000-fold in anaerobic glucose-fermenting cultures and showed further improvement under fumarate-respiring conditions. An important bottleneck to overcome appears to be product excretion, based on much higher recorded intracellular concentrations of benzylsuccinate, compared to those excreted. While no export system is known for benzylsuccinate, we observed an increased product yield after adding an unspecific mechanosensitive channel to the constructed pathway. [ABSTRACT FROM AUTHOR]

α-Methylacyl-CoA racemase in M. tuberculosis (MCR) has an essential role in fatty acid metabolism and cholesterol utilization, contributing to the bacterium’s survival and persistence. Understanding the enzymatic activity and structural features of MCR provides insights into its physiological and pathological significance and potential as a therapeutic target. Here, we report high-resolution crystal structures for wild-type MCR in a new crystal form (at 1.65 Å resolution) and for three active-site mutants, H126A, D156A and E241A, at 2.45, 1.64 and 1.85 Å resolutions, respectively. Our analysis of the new wild-type structure revealed a similar dimeric arrangement of MCR molecules to that previously reported and details of the catalytic site. The determination of the structures of these H126A, D156A and E241A mutants, along with their detailed kinetic analysis, has now allowed for a rigorous assessment of their catalytic properties. No significant change outside the enzymatic active site was observed in the three mutants, establishing that the diminution of catalytic activity is mainly attributable to disruption of the catalytic apparatus involving key hydrogen bonding and water-mediated interactions. The wild-type structure, together with detailed mutational and biochemical data, provide a basis for understanding the catalytic properties of this enzyme, which is important for the design of future anti-tuberculosis drug molecules.

(R)-Benzylsuccinate is generated in anaerobic toluene degradation by the radical addition of toluene to fumarate and further degraded to benzoyl-CoA by a β-oxidation pathway. Using metabolic modules for benzoate transport and activation to benzoyl-CoA and the enzymes of benzylsuccinate β-oxidation, we established an artificial pathway for benzylsuccinate production in Escherichia coli, which is based on its degradation pathway running in reverse. Benzoate is supplied to the medium but needs to be converted to benzoyl-CoA by an uptake transporter and a benzoate-CoA ligase or CoA-transferase. In contrast, the second substrate succinate is endogenously produced from glucose under anaerobic conditions, and the constructed pathway includes a succinyl-CoA:benzylsuccinate CoA-transferase that activates it to the CoA-thioester. We present first evidence for the feasibility of this pathway and explore product yields under different growth conditions. Compared to aerobic cultures, the product yield increased more than 1000-fold in anaerobic glucose-fermenting cultures and showed further improvement under fumarate-respiring conditions. An important bottleneck to overcome appears to be product excretion, based on much higher recorded intracellular concentrations of benzylsuccinate, compared to those excreted. While no export system is known for benzylsuccinate, we observed an increased product yield after adding an unspecific mechanosensitive channel to the constructed pathway.

Clostridium beijerinckii CC101 was engineered to overexpress aldehyde/alcohol dehydrogenase ( adh E2) and CoA-transferase ( ctf AB). Solvent production and acid assimilation were compared between the parental and engineered strains expressing only adh E2 (CC101-SV4) and expressing adh E2, ald and ctfAB (CC101-SV6). CC101-SV4 showed an early butanol production from glucose but stopped pre-maturely at a low butanol concentration of ∼6 g/L. Compared to CC101, CC101-SV6 produced more butanol (∼12 g/L) from glucose and was able to re-assimilate more acids, which prevented “acid crash” and increased butanol production, under all conditions studied. CC101-SV6 also showed better ability in using glucose and xylose present in sugarcane bagasse hydrolysate, and produced 9.4 g/L solvents (acetone, butanol and ethanol) compared to only 2.6 g/L by CC101, confirming its robustness and better tolerance to hydrolysate inhibitors. The engineered strain of C. beijerinckii overexpressing adh E2 and ctf AB should have good potential for producing butanol from lignocellulosic biomass hydrolysates. [ABSTRACT FROM AUTHOR]

Abstract: Formation and excretion of acetate as a metabolic end product of energy metabolism occurs in many protist and helminth parasites, such as the parasitic helminths Fasciola hepatica, Haemonchus contortus and Ascaris suum, and the protist parasites, Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis as well as Trypanosoma and Leishmania spp. In all of these parasites acetate is a main end product of their energy metabolism, whereas acetate formation does not occur in their mammalian hosts. Acetate production might therefore harbour novel targets for the development of new anti-parasitic drugs. In parasites, acetate is produced from acetyl-CoA by two different reactions, both involving substrate level phosphorylation, that are catalysed by either a cytosolic acetyl-CoA synthetase (ACS) or an organellar acetate:succinate CoA-transferase (ASCT). The ACS reaction is directly coupled to ATP synthesis, whereas the ASCT reaction yields succinyl-CoA for ATP formation via succinyl-CoA synthetase (SCS). Based on recent work on the ASCTs of F. hepatica, T. vaginalis and Trypanosoma brucei we suggest the existence of three subfamilies of enzymes within the CoA-transferase family I. Enzymes of these three subfamilies catalyse the ASCT reaction in eukaryotes via the same mechanism, but the subfamilies share little sequence homology. The CoA-transferases of the three subfamilies are all present inside ATP-producing organelles of parasites, those of subfamily IA in the mitochondria of trypanosomatids, subfamily IB in the mitochondria of parasitic worms and subfamily IC in hydrogenosome-bearing parasites. Together with the recent characterisation among non-parasitic protists of yet a third route of acetate formation involving acetate kinase (ACK) and phosphotransacetylase (PTA) that was previously unknown among eukaryotes, these recent developments provide a good opportunity to have a closer look at eukaryotic acetate formation. [Copyright &y& Elsevier]

4-Hydroxybutyrate CoA-transferases (4-HB-CoAT) takes part in the fermentation of 4-aminobutyrate to ammonia, acetate, and butyrate in anaerobic bacteria such as Clostridium aminobutyricum and Porphyromonas gingivalis or facultative anaerobic bacteria such as Shewanella oneidensis. Site-directed mutagenesis of the highly active enzyme has identified the catalytic glutamate residue as E238. Crystal structure of this enzyme has been determined at a resolution of 1.85 Å. The 438-amino acid residue polypeptide chain folds into two topologically similar domains with an open α/β-fold, which is also found in other CoAT family I and family II members. The data indicate that the members of CoAT families I and II are closely related; the latter only lacking the catalytic glutamate residue. A putative co-substrate binding site for the 4-HB-CoAT was identified, in which a 4-hydroxybutyrate molecule has been modeled. This site is also responsible for binding the acetyl group of acetyl-CoA or the succinyl group of succinyl-CoA in succinyl-CoA:3-oxoacid CoA-transferase from mammalian mitochondria. Mutations of relevant active site amino acid residues have been produced and their activities tested to corroborate the proposed structural model for substrate binding. 4-HB-CoAT from C. aminobutyricum represents the only functionally characterized 4-HB-CoAT present in the structural database. [ABSTRACT FROM AUTHOR]

Abstract: Saccharomyces cerevisiae and Neurospora crassa mutants defective in the so-called acetyl-CoA hydrolases Ach1p and Acu-8, respectively, display a severe growth defect on acetate, which is most strongly pronounced under acidic conditions. Acetyl-CoA hydrolysis is an energy wasting process and therefore denoted as a biochemical conundrum. Acetyl-CoA hydrolases show high sequence identity to the CoA-transferase CoaT from Aspergillus nidulans. Therefore, we extensively re-characterised the yeast enzyme. Ach1p showed highest specific activity for the CoASH transfer from succinyl-CoA to acetate and only a minor acetyl-CoA-hydrolase activity. Complementation of an ach1 mutant with the coaT gene reversed the growth defect on acetate confirming the in vivo function of Ach1p as a CoA-transferase. Our results imply that Ach1p is involved in mitochondrial acetate detoxification by a CoASH transfer from succinyl-CoA to acetate. Thereby, Ach1p does not perform the energy wasting hydrolysis of acetyl-CoA but conserves energy by the detoxification of mitochondrial acetate. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: α-Methylacyl-CoA racemases are essential enzymes for branched-chain fatty acid metabolism. Their reaction mechanism and the structural basis of their wide substrate specificity are poorly understood. High-resolution crystal structures of Mycobacterium tuberculosis α-methylacyl-CoA racemase (MCR) complexed with substrate molecules show the active site geometry required for catalysis of the interconversion of (2S) and (2R)-methylacyl-CoA. The thioester oxygen atom and the 2-methyl group are in a cis-conformation with respect to each other. The thioester oxygen atom fits into an oxyanion hole and the 2-methyl group points into a hydrophobic pocket. The active site geometry agrees with a 1,1-proton transfer mechanism in which the acid/base-pair residues are His126 and Asp156. The structures of the complexes indicate that the acyl chains of the S-substrate and the R-substrate bind in an S-pocket and an R-pocket, respectively. A unique feature of MCR is a large number of methionine residues in the acyl binding region, located between the S-pocket and the R-pocket. It appears that the (S) to (R) interconversion of the 2-methylacyl chiral center is coupled to a movement of the acyl group over this hydrophobic, methionine-rich surface, when moving from its S-pocket to its R-pocket, whereas the 2-methyl moiety and the CoA group remain fixed in their respective pockets. [Copyright &y& Elsevier]

The fermentation ofβ-alanine byClostridium propionicumproceeds via activation to the CoA-thiol ester, followed by deamination to acryloyl-CoA, which is also an intermediate in the fermentation ofl-alanine. By shifting the organism from the carbon and energy sourceα-alanine toβ-alanine, the enzymeβ-alanyl-CoA:ammonia lyase is induced 300-fold (≈ 30% of the soluble protein). The low basal lyase activity is encoded by theacl1gene, whereas the almost identicalacl2gene (six amino acid substitutions) is responsible for the high activity after growth onβ-alanine. The deducedβ-alanyl-CoA:ammonia lyase proteins are related to putativeβ-aminobutyryl-CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria.β-Alanyl-CoA:ammonia lyase 2 was purified to homogeneity and characterized as a heteropentamer composed of 16 kDa subunits. The apparentKm value for acryloyl-CoA was measured as 23 ± 4 µm, independent of the concentration of the second substrate ammonia;kcat/ Km was calculated as 107 m−1·s−1. The apparentKm for ammonia was much higher, 70 ± 5 mmat 150 µmacryloyl-CoA with a much lowerkcat/ Km of 4 × 103 m−1·s−1. In the reverse reaction, aKm of 210 ± 30 µM was obtained forβ-alanyl-CoA. The elimination of ammonia was inhibited by 70% at 100 mmammonium chloride. The content ofβ-alanyl-CoA:ammonia lyase inβ-alanine grown cells is about 100 times higher than that required to sustain the growth rate of the organism. It is therefore suggested that the enzyme is needed to bind acryloyl-CoA, in order to keep the toxic free form at a very low level. A formula was derived for the calculation of isomerization equilibra betweenl-alanine/β-alanine ord-lactate/3-hydroxypropionate. [ABSTRACT FROM AUTHOR]

Embryos and larvae of the brine shrimp, Artemia franciscana, were shown previously to possess a protein, now termed p49, which cross-links microtubules in vitro. Molecular characteristics of p49 were described, but the protein's identity and its role in the cell were not determined. Degenerate oligonucleotide primers designed on the basis of peptide sequence obtained by Edman degradation during this study were used to generate p49 cDNAs by RT-PCR and these were cloned and sequenced. Comparison with archived sequences revealed that the deduced amino acid sequence of p49 resembled the Drosophila gene product CG7920, as well as related proteins encoded in the genomes of Anopheles and Caenorhabditis. Similar proteins exist in several bacteria but no evident homologues were found in vertebrates and plants, and only very distant homologues resided in yeast. When evolutionary relationships were compared, p49 and the homologues from Drosophila, Anopheles and Caenorhabditis formed a distinct subcluster within phylogenetic trees. Additionally, the predicted secondary structures of p49, 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum and glutaconate CoA-transferase from Acidaminococcus fermentans were similar and the enzymes may possess related catalytic mechanisms. The purified Artemia protein exhibited 4-hydroxybutyrate CoA-transferase activity, thereby establishing p49 as the first crustacean CoA-transferase to be characterized. Probing of Western blots with an antibody against p49 revealed a cross-reactive protein in Drosophila that associated with microtubules, but to a lesser extent than did p49 from Artemia. [ABSTRACT FROM AUTHOR]

Propionate CoA-transferase from Clostridium propionicum has been purified and the gene encoding the enzyme has been cloned and sequenced. The enzyme was rapidly and irreversibly inactivated by sodium borohydride or hydroxylamine in the presence of propionyl-CoA. The reduction of the thiol ester between a catalytic site glutamate and CoA with borohydride and the cleavage by hydroxylamine were used to introduce a site-specific label, which was followed by␣MALDI-TOF-MS. This allowed the identification of glutamate 324 at the active site. Propionate CoA-transferase and similar proteins deduced from the genomes of Escherichia coli , Staphylococcus aureus , Bacillus halodurans and Aeropyrum pernix are proposed to form a novel subclass of CoA-transferases. Secondary structure element predictions were generated and compared to known crystal structures in the databases. A high degree of structural similarity was observed between the arrangement of secondary structure elements in these proteins and glutaconate CoA-transferase from Acidaminococcus fermentans . [ABSTRACT FROM AUTHOR]

Phenyllactate dehydratase from Clostridium sporogenes grown anaerobically on l-phenylalanine catalyses the reversible syn-dehydration of (R)-phenyllactate to (E)-cinnamate. Purification yielded a heterotrimeric enzyme complex (130 ± 15 kDa) composed of FldA (46 kDa), FldB (43 kDa) and FldC (40 kDa). By re-chromatography on Q-Sepharose, the major part of FldA could be separated and identified as oxygen insensitive cinnamoyl-CoA:phenyllactate CoA-transferase, whereas the transferase depleted trimeric complex retained oxygen sensitive phenyllactate dehydratase activity and contained about one [4Fe-4S] cluster. The dehydratase activity required 10 µm FAD, 0.4 mm ATP, 2.5 mm MgCl2, 0.1 mm NADH, 5 µm cinnamoyl-CoA and small amounts of cell-free extract (10 µg protein per mL) similar to that known for 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. The N-terminus of the homogenous FldA (39 amino acids) is homologous to that of CaiB (39% sequence identity) involved in carnitine metabolism in Escherichia coli. Both enzymes are members of an emerging group of CoA-transferases which exhibit high substrate specificity but apparently do not form enzyme CoA-ester intermediates. It is concluded that dehydration of (R)-phenyllactate to (E)-cinnamate proceeds in two steps, a CoA-transfer from cinnamoyl-CoA to phenyllactate, catalysed by FldA, followed by the dehydration of phenyllactyl-CoA, catalysed by FldB and FldC, whereby the noncovalently bound prosthetic group cinnamoyl-CoA is regenerated. This demonstrates the necessity of a 2-hydroxyacyl-CoA intermediate in the dehydration of 2-hydroxyacids. The transient CoA-ester formation during the dehydration of phenyllactate resembles that during citrate cleavage catalysed by bacterial citrate lyase, which contain a derivative of acetyl-CoA covalently bound to an acyl-carrier-protein (ACP). [ABSTRACT FROM AUTHOR]

Coenzyme A transferases (CoATs) are important enzymes involved in carbon chain elongation, contributing to medium-chain fatty acid (MCFA) biosynthesis. For example, butyryl-CoA:acetate CoA transferase (BCoAT) is responsible for the final step of butyrate synthesis from butyryl-CoA. However, little is known about caproyl-CoA:acetate CoA-transferase (CCoAT), which is responsible for the final step of caproate synthesis from caproyl-CoA. In the present study, two CoAT genes from Ruminococcaceae bacterium CPB6 and Clostridium tyrobutyricum BEY8 were identified by gene cloning and expression analysis. Enzyme assays and kinetic studies were carried out using butyryl-CoA or caproyl-CoA as the substrate. CPB6-CoAT can catalyze the conversion of both butyryl-CoA into butyrate and caproyl-CoA into caproate, but its catalytic efficiency with caproyl-CoA as the substrate was 3.8-times higher than that with butyryl-CoA. In contrast, BEY8-CoAT had only BCoAT activity, not CCoAT activity. This demonstrated the existence of a specific CCoAT involved in chain elongation via the reverse β-oxidation pathway. Comparative bioinformatics analysis showed the presence of a highly conserved motif (GGQXDFXXGAXX) in CoATs, which is predicted to be the active center. Single point mutations in the conserved motif of CPB6-CoAT (Asp346 and Ala351) led to marked decreases in the activity for butyryl-CoA and caproyl-CoA, indicating that the conserved motif is the active center of CPB6-CoAT and that Asp346 and Ala351 have a significant impact on the enzymatic activity. This work provides insight into the function of CCoAT in caproic acid biosynthesis and improves understanding of the chain elongation pathway for MCFA production., (© 2021 The Author(s).)