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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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Full-length sequence alignment of three enoyl-ACP reductase enzymes from different organisms.The sequences are from V. cholerae, B. mallei and X. oryzae. Y226, Y236 and K245 of xoFabV and their corresponding residues in the other two enzymes are labelled with asterisks. V246 of xoFabV and its corresponding residues are labelled with a colon. The sequence alignment was performed using T-Coffee, and the figure was made using ESPript [34].
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pone-0026743-g005: Full-length sequence alignment of three enoyl-ACP reductase enzymes from different organisms.The sequences are from V. cholerae, B. mallei and X. oryzae. Y226, Y236 and K245 of xoFabV and their corresponding residues in the other two enzymes are labelled with asterisks. V246 of xoFabV and its corresponding residues are labelled with a colon. The sequence alignment was performed using T-Coffee, and the figure was made using ESPript [34].

Mentions: Although no FabV structure has been reported, previous bioinformatic and mutagenesis studies of bmFabV suggested that in addition to the conserved tyrosine (Y235) and the lysine (K244) residues of the Y-X8-K motif, an adjacent lysine (K245) in bmFabV also plays an important role in substrate binding. However, a sequence alignment of vcFabV (YP_001217283.2), bmFabV (YP_102617.1) and xoFabV using T-Coffee [20], [21] revealed that K245 present in bmFabV (Fig. 5) is altered to a non-polar valine (V246) in xoFabV.


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)

Full-length sequence alignment of three enoyl-ACP reductase enzymes from different organisms.The sequences are from V. cholerae, B. mallei and X. oryzae. Y226, Y236 and K245 of xoFabV and their corresponding residues in the other two enzymes are labelled with asterisks. V246 of xoFabV and its corresponding residues are labelled with a colon. The sequence alignment was performed using T-Coffee, and the figure was made using ESPript [34].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026743-g005: Full-length sequence alignment of three enoyl-ACP reductase enzymes from different organisms.The sequences are from V. cholerae, B. mallei and X. oryzae. Y226, Y236 and K245 of xoFabV and their corresponding residues in the other two enzymes are labelled with asterisks. V246 of xoFabV and its corresponding residues are labelled with a colon. The sequence alignment was performed using T-Coffee, and the figure was made using ESPript [34].
Mentions: Although no FabV structure has been reported, previous bioinformatic and mutagenesis studies of bmFabV suggested that in addition to the conserved tyrosine (Y235) and the lysine (K244) residues of the Y-X8-K motif, an adjacent lysine (K245) in bmFabV also plays an important role in substrate binding. However, a sequence alignment of vcFabV (YP_001217283.2), bmFabV (YP_102617.1) and xoFabV using T-Coffee [20], [21] revealed that K245 present in bmFabV (Fig. 5) is altered to a non-polar valine (V246) in xoFabV.

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