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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 - PLoS ONE (2011)

Bottom Line: Enoyl-ACP reductase (ENR) catalyses the last reduction reaction in the fatty acid elongation cycle in bacteria and is a good antimicrobial target candidate.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.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.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People's Republic of China.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

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Progress curve analysis of the wild-type and mutant xoFabV variants in the NADH oxidation assay.The enzyme activity of wild-type and mutant xoFabV was determined by monitoring the oxidation of NADH to NAD+ at 340 nm. The reaction was initiated by adding the substrate crotonyl-CoA and was monitored for 10 min at 25°C.
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pone-0026743-g007: Progress curve analysis of the wild-type and mutant xoFabV variants in the NADH oxidation assay.The enzyme activity of wild-type and mutant xoFabV was determined by monitoring the oxidation of NADH to NAD+ at 340 nm. The reaction was initiated by adding the substrate crotonyl-CoA and was monitored for 10 min at 25°C.

Mentions: The expression of xoFabV mutants D111A, Y236A, Y236F, K245A and K245R in the E. coli fabI (Ts) strain JP1111 [2] did not restore in vivo fatty acid synthesis at the non-permissive temperature of 42°C (Fig. 6). Mutant Y53A partly restored fatty acid synthesis, while S50A, Y53F, F113A, Y226F, V246A and T276A restored synthesis to the wild-type level. NADH oxidation was assayed in vitro with the model substrate crotonyl-CoA. Kinetic parameters were measured only for wild-type xoFabV. The values of kcat, Km,NADH and Km,Crotonyl-CoA were 1335±145 min−1, 18.7±2.0 µM and 293±16.5 µM, respectively (Table 3), indicating that xoFabV has a similar catalytic efficiency for crotonyl-CoA compared to bmFabV [12]. For the xoFabV mutants, only progress curves were measured (Fig. 7). The curves showed that the enzymatic activities of mutants Y53A, D111A, Y236A, Y236F, K245A, K245R and T276A were not detected in the NADH oxidation assay; Y53F and Y226F catalysed the reduction at about 50% efficiency; F113A had a speed that was one sixth that of the wild-type and mutants S50A and V246A were as efficient as the wild-type enzyme.


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 - PLoS ONE (2011)

Progress curve analysis of the wild-type and mutant xoFabV variants in the NADH oxidation assay.The enzyme activity of wild-type and mutant xoFabV was determined by monitoring the oxidation of NADH to NAD+ at 340 nm. The reaction was initiated by adding the substrate crotonyl-CoA and was monitored for 10 min at 25°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198815&req=5

pone-0026743-g007: Progress curve analysis of the wild-type and mutant xoFabV variants in the NADH oxidation assay.The enzyme activity of wild-type and mutant xoFabV was determined by monitoring the oxidation of NADH to NAD+ at 340 nm. The reaction was initiated by adding the substrate crotonyl-CoA and was monitored for 10 min at 25°C.
Mentions: The expression of xoFabV mutants D111A, Y236A, Y236F, K245A and K245R in the E. coli fabI (Ts) strain JP1111 [2] did not restore in vivo fatty acid synthesis at the non-permissive temperature of 42°C (Fig. 6). Mutant Y53A partly restored fatty acid synthesis, while S50A, Y53F, F113A, Y226F, V246A and T276A restored synthesis to the wild-type level. NADH oxidation was assayed in vitro with the model substrate crotonyl-CoA. Kinetic parameters were measured only for wild-type xoFabV. The values of kcat, Km,NADH and Km,Crotonyl-CoA were 1335±145 min−1, 18.7±2.0 µM and 293±16.5 µM, respectively (Table 3), indicating that xoFabV has a similar catalytic efficiency for crotonyl-CoA compared to bmFabV [12]. For the xoFabV mutants, only progress curves were measured (Fig. 7). The curves showed that the enzymatic activities of mutants Y53A, D111A, Y236A, Y236F, K245A, K245R and T276A were not detected in the NADH oxidation assay; Y53F and Y226F catalysed the reduction at about 50% efficiency; F113A had a speed that was one sixth that of the wild-type and mutants S50A and V246A were as efficient as the wild-type enzyme.

Bottom Line: Enoyl-ACP reductase (ENR) catalyses the last reduction reaction in the fatty acid elongation cycle in bacteria and is a good antimicrobial target candidate.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.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.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People's Republic of China.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Show MeSH
Related in: MedlinePlus