Publications

• Mitochondrial trans-2-enoyl-CoA reductase of wax ester fermentation from Euglena gracilis defines a new family of enzymes involved in lipid synthesis.

  Hoffmeister M., Piotrowski M., Nowitzki U., Martin W.

  Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is tr ... >> More

  Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catalyzes reduction of enoyl-CoA to acyl-CoA. Trans-2-enoyl-CoA reductase from Euglena was purified 1700-fold to electrophoretic homogeneity and was active with NADH and NADPH as the electron donor. The active enzyme is a monomer with molecular mass of 44 kDa. The amino acid sequence of tryptic peptides determined by electrospray ionization mass spectrometry were used to clone the corresponding cDNA, which encoded a polypeptide that, when expressed in Escherichia coli and purified by affinity chromatography, possessed trans-2-enoyl-CoA reductase activity close to that of the enzyme purified from Euglena. Trans-2-enoyl-CoA reductase activity is present in mitochondria and the mRNA is expressed under aerobic and anaerobic conditions. Using NADH, the recombinant enzyme accepted crotonyl-CoA (km=68 microm) and trans-2-hexenoyl-CoA (km=91 microm). In the crotonyl-CoA-dependent reaction, both NADH (km=109 microm) or NADPH (km=119 microm) were accepted, with 2-3-fold higher specific activities for NADH relative to NADPH. Trans-2-enoyl-CoA reductase homologues were not found among other eukaryotes, but are present as hypothetical reading frames of unknown function in sequenced genomes of many proteobacteria and a few Gram-positive eubacteria, where they occasionally occur next to genes involved in fatty acid and polyketide biosynthesis. Trans-2-enoyl-CoA reductase assigns a biochemical activity, NAD(P)H-dependent acyl-CoA synthesis from enoyl-CoA, to one member of this gene family of previously unknown function. << Less

  J. Biol. Chem. 280:4329-4338(2005) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Determination of the crystal structure and active residues of FabV, the enoyl-ACP reductase from Xanthomonas oryzae.

  Li H., Zhang X., Bi L., He J., Jiang T.

  <h4>Background</h4>Enoyl-ACP reductase (ENR) catalyses the last reduction reaction in the fatty acid elongation cycle in bacteria and is a good antimicrobial target candidate. FabV is the most recently discovered class of ENR, but we lack information about the atomic structure and the key residues ... >> More

  <h4>Background</h4>Enoyl-ACP reductase (ENR) catalyses the last reduction reaction in the fatty acid elongation cycle in bacteria and is a good antimicrobial target candidate. FabV is the most recently discovered class of ENR, but we lack information about the atomic structure and the key residues involved in reductase activity except for the known conserved tyrosine and lysine residues in the Y-X(8)-K active site motif.<h4>Methodology/principal findings</h4>Here we report the crystal structure of FabV from Xanthomonas oryzae (xoFabV). The crystal structure of this enzyme has been solved to 1.6 Å resolution in space group P2(1)2(1)2(1). The model of xoFabV consists of one monomer in the asymmetric unit which is composed of 13 α-helices and 11 β-strands, representing a canonical Rossmann fold architecture. Structural comparison presents that the locations of the conserved tyrosine (Y236) and lysine (K245) residues in the Y-X(8)-K active site motif of xoFabV and the Y-X(6)-K motif of ecFabI are notably similar. However, the conformations of Y236 in xoFabV and Y156 in ecFabI are distinct. Structure-based site-directed mutagenesis and enzymatic activity assays reveal that in addition to the conserved Y236 and K245 in the Y-X(8)-K motif, Y53, D111 and Y226 are key residues implicated in the reductase activity, and F113 and T276 are also important for enzyme function. Moreover, a proposed active lysine located immediately after the Y-X(8)-K motif in FabV from Burkholderia mallei (bmFabV) is altered to an inactive V246 in xoFabV.<h4>Conclusions/significance</h4>We determine the first crystal structure of the FabV enzyme and identify several residues important for its enzymatic activity. These findings lay a solid foundation for the development of specific antibacterial inhibitors of the pathogenic bacteria, such as Vibrio cholerae, Burkholderia species and Xanthomonas species. << Less

  PLoS ONE 6:E26743-E26743(2011) [PubMed] [EuropePMC]

• A novel prokaryotic trans-2-enoyl-CoA reductase from the spirochete Treponema denticola.

  Tucci S., Martin W.

  An NADH-dependent trans-2-enoyl-CoA reductase (EC1.1.1.36) from the Gram negative spirochete Treponema denticola was identified, expressed and biochemically characterized. The recombinant protein is a monomeric enzyme with a molecular mass of 44 kDa with a specific activity of 43+/-4.8 U/mg (micro ... >> More

  An NADH-dependent trans-2-enoyl-CoA reductase (EC1.1.1.36) from the Gram negative spirochete Treponema denticola was identified, expressed and biochemically characterized. The recombinant protein is a monomeric enzyme with a molecular mass of 44 kDa with a specific activity of 43+/-4.8 U/mg (micromol mg(-1)min(-1)) and K(m) value of 2.7 microM for crotonoyl-CoA. This NADH-dependent trans-2-enoyl-CoA reductase represents the first enzymatically characterized member of a prokaryotic protein family involved in a fatty acid synthesis pathway that is distinct from the familiar fatty acid synthase system. << Less

  FEBS Lett. 581:1561-1566(2007) [PubMed] [EuropePMC]

• Biochemical and structural characterization of the trans-enoyl-CoA reductase from Treponema denticola.

  Bond-Watts B.B., Weeks A.M., Chang M.C.

  The production of fatty acids is an important cellular pathway for both cellular function and the development of engineered pathways for the synthesis of advanced biofuels. Despite the conserved reaction chemistry of various fatty acid synthase systems, the individual isozymes that catalyze these ... >> More

  The production of fatty acids is an important cellular pathway for both cellular function and the development of engineered pathways for the synthesis of advanced biofuels. Despite the conserved reaction chemistry of various fatty acid synthase systems, the individual isozymes that catalyze these steps are quite diverse in their structural and biochemical features and are important for controlling differences at the cellular level. One of the key steps in the fatty acid elongation cycle is the enoyl-ACP (CoA) reductase function that drives the equilibrium forward toward chain extension. In this work, we report the structural and biochemical characterization of the trans-enoyl-CoA reductase from Treponema denticola (tdTer), which has been utilized for the engineering of synthetic biofuel pathways with an order of magnitude increase in product titers compared to those of pathways constructed with other enoyl-CoA reductase components. The crystal structure of tdTer was determined to 2.00 Å resolution and shows that the Ter enzymes are distinct from members of the FabI, FabK, and FabL families but are highly similar to members of the FabV family. Further biochemical studies show that tdTer uses an ordered bi-bi mechanism initiated by binding of the NADH redox cofactor, which is consistent with the behavior of other enoyl-ACP (CoA) reductases. Mutagenesis of the substrate binding loop, characterization of enzyme activity with respect to crotonyl-CoA, hexenoyl-CoA, and dodecenoyl-CoA substrates, and product inhibition by lauroyl-CoA suggest that this region is important for controlling chain length specificity, with the major portal playing a more important role for longer chain length substrates. << Less

  Biochemistry 51:6827-6837(2012) [PubMed] [EuropePMC]

• Triclosan resistance of Pseudomonas aeruginosa PAO1 is due to FabV, a triclosan-resistant enoyl-acyl carrier protein reductase.

  Zhu L., Lin J., Ma J., Cronan J.E., Wang H.

  Triclosan, a very widely used biocide, specifically inhibits fatty acid synthesis by inhibition of enoyl-acyl carrier protein (ACP) reductase. Escherichia coli FabI is the prototypical triclosan-sensitive enoyl-ACP reductase, and E. coli is extremely sensitive to the biocide. However, other bacter ... >> More

  Triclosan, a very widely used biocide, specifically inhibits fatty acid synthesis by inhibition of enoyl-acyl carrier protein (ACP) reductase. Escherichia coli FabI is the prototypical triclosan-sensitive enoyl-ACP reductase, and E. coli is extremely sensitive to the biocide. However, other bacteria are resistant to triclosan, because they encode triclosan-resistant enoyl-ACP reductase isozymes. In contrast, the triclosan resistance of Pseudomonas aeruginosa PAO1 has been attributed to active efflux of the compound (R. Chuanchuen, R. R. Karkhoff-Schweizer, and H. P. Schweizer, Am. J. Infect. Control 31:124-127, 2003). We report that P. aeruginosa contains two enoyl-ACP reductase isozymes, the previously characterized FabI homologue plus a homologue of FabV, a triclosan-resistant enoyl-ACP reductase recently demonstrated in Vibrio cholerae. By deletion of the genes encoding P. aeruginosa FabI and FabV, we demonstrated that FabV confers triclosan resistance on P. aeruginosa. Upon deletion of the fabV gene, the mutant strain became extremely sensitive to triclosan (>2,000-fold more sensitive than the wild-type strain), whereas the mutant strain lacking FabI remained completely resistant to the biocide. << Less

  Antimicrob. Agents Chemother. 54:689-698(2010) [PubMed] [EuropePMC]

• Structures of trans-2-enoyl-CoA reductases from Clostridium acetobutylicum and Treponema denticola: insights into the substrate specificity and the catalytic mechanism.

  Hu K., Zhao M., Zhang T., Zha M., Zhong C., Jiang Y., Ding J.

  TERs (trans-2-enoyl-CoA reductases; EC 1.3.1.44), which specifically catalyse the reduction of crotonyl-CoA to butyryl-CoA using NADH as cofactor, have recently been applied in the design of robust synthetic pathways to produce butan-1-ol as a biofuel. We report in the present paper the characteri ... >> More

  TERs (trans-2-enoyl-CoA reductases; EC 1.3.1.44), which specifically catalyse the reduction of crotonyl-CoA to butyryl-CoA using NADH as cofactor, have recently been applied in the design of robust synthetic pathways to produce butan-1-ol as a biofuel. We report in the present paper the characterization of a CaTER (a TER homologue in Clostridium acetobutylicum), the structures of CaTER in apo form and in complexes with NADH and NAD+, and the structure of TdTER (Treponema denticola TER) in complex with NAD+. Structural and sequence comparisons show that CaTER and TdTER share approximately 45% overall sequence identity and high structural similarities with the FabV class enoyl-acyl carrier protein reductases in the bacterial fatty acid synthesis pathway, suggesting that both types of enzymes belong to the same family. CaTER and TdTER function as monomers and consist of a cofactor-binding domain and a substrate-binding domain with the catalytic active site located at the interface of the two domains. Structural analyses of CaTER together with mutagenesis and biochemical data indicate that the conserved Glu75 determines the cofactor specificity, and the conserved Tyr225, Tyr235 and Lys244 play critical roles in catalysis. Upon cofactor binding, the substrate-binding loop changes from an open conformation to a closed conformation, narrowing a hydrophobic channel to the catalytic site. A modelling study shows that the hydrophobic channel is optimal in both width and length for the binding of crotonyl-CoA. These results provide molecular bases for the high substrate specificity and the catalytic mechanism of TERs. << Less

  Biochem. J. 449:79-89(2013) [PubMed] [EuropePMC]

• Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase.

  Massengo-Tiasse R.P., Cronan J.E.

  Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia coli that is the target of the antibacterial compound, triclosan. However, some Gram-positive bacteria are naturally resistant ... >> More

  Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia coli that is the target of the antibacterial compound, triclosan. However, some Gram-positive bacteria are naturally resistant to triclosan due to the presence of the triclosan-resistant enoyl-ACP reductase isoforms, FabK and FabL. The genome of the Gram-negative bacterium, Vibrio cholerae lacks a gene encoding a homologue of any of the three known enoyl-ACP reductase isozymes suggesting that this organism encodes a novel fourth enoyl-ACP reductase isoform. We report that this is the case. The gene encoding the new isoform, called FabV, was isolated by complementation of a conditionally lethal E. coli fabI mutant strain and was shown to restore fatty acid synthesis to the mutant strain both in vivo and in vitro. Like FabI and FabL, FabV is a member of the short chain dehydrogenase reductase superfamily, although it is considerably larger (402 residues) than either FabI (262 residues) or FabL (250 residues). The FabV, FabI and FabL sequences can be aligned, but only poorly. Alignment requires many gaps and yields only 15% identical residues. Thus, FabV defines a new class of enoyl-ACP reductase. The native FabV protein has been purified to homogeneity and is active with both crotonyl-ACP and the model substrate, crotonyl-CoA. In contrast to FabI and FabL, FabV shows a very strong preference for NADH over NADPH. Expression of FabV in E. coli results in markedly increased resistance to triclosan and the purified enzyme is much more resistant to triclosan than is E. coli FabI. << Less

  J. Biol. Chem. 283:1308-1316(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Mechanism and inhibition of the FabV enoyl-ACP reductase from Burkholderia mallei.

  Lu H., Tonge P.J.

  Enoyl-ACP reductases catalyze the final step in the elongation cycle of the bacterial fatty acid biosynthesis (FAS-II) pathway. At present, four distinct enoyl-ACP reductases have been identified, which are the products of the fabI, fabL, fabK, and fabV genes. The FabV enoyl-ACP reductase is the m ... >> More

  Enoyl-ACP reductases catalyze the final step in the elongation cycle of the bacterial fatty acid biosynthesis (FAS-II) pathway. At present, four distinct enoyl-ACP reductases have been identified, which are the products of the fabI, fabL, fabK, and fabV genes. The FabV enoyl-ACP reductase is the most recent member of this enzyme class and was originally identified in Vibrio cholerae by Cronan and co-workers [Massengo-Tiasse, R. P., and Cronan, J. E. (2008) Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase. J. Biol. Chem. 283, 1308-1316]. In this work, a detailed kinetic analysis of the mechanism of the FabV enzyme from Burkholderia mallei (bmFabV) has been undertaken, which reveals that bmFabV catalyzes a sequential bi-bi mechanism with NADH binding first and NAD(+) dissociating last. The enzyme is a member of the short chain dehydrogenase/reductase superfamily in which the catalytic tyrosine (Y235) and lysine (K244) residues are organized in the consensus Tyr-(Xaa)(8)-Lys motif. The role of these active site residues has been investigated using site-directed mutagenesis which has shown that both Y235 and K244 are involved in acid-base chemistry during substrate reduction. Sequence alignment and site-directed mutagenesis also identify a second lysine in the active site (K245) that has an important role in binding of the enoyl substrate. Because of interests in developing inhibitors of bmFabV, a detailed analysis of the inhibition of the enzyme by triclosan has been conducted showing that triclosan is a competitive inhibitor with respect to NADH and an uncompetitive inhibitor with respect to the substrate 2-dodecenoyl-CoA (K(i) = 0.4 muM). In combination with fluorescence binding experiments, we conclude that triclosan binds to the enzyme-NAD(+) product complex which is in rapid and reversible equilibrium with other intermediates on the reaction pathway. << Less

  Biochemistry 49:1281-1289(2010) [PubMed] [EuropePMC]