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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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Crystal structure of xoFabV.The structure consists of 13 α-helices and 11 β-strands, representing a classic Rossmann fold architecture. The secondary structures are shown in different colours and are labelled with the corresponding numbers. (A) Side view. (B) Top view. Figures 1–4 were made using PyMOL (DeLano Scientific, Palo Alto, California, USA; http://www.pymol.org).
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pone-0026743-g001: Crystal structure of xoFabV.The structure consists of 13 α-helices and 11 β-strands, representing a classic Rossmann fold architecture. The secondary structures are shown in different colours and are labelled with the corresponding numbers. (A) Side view. (B) Top view. Figures 1–4 were made using PyMOL (DeLano Scientific, Palo Alto, California, USA; http://www.pymol.org).

Mentions: The SeMet-xoFabV crystal contains one monomer in the asymmetric unit. The density map was of high quality and allowed us to build an almost complete model containing 390 residues (C12-L401) but lacking the His tag and thrombin cleavage site at the N-terminus and A402. Each xoFabV monomer consists of 13 α-helices (α1 – α13) and 11 β-strands (β1 – β11) (Fig. 1). Strands β1 – β4 and β9 – 11 form a parallel β-sheet located at the centre of the structure. This β-sheet is flanked on one side by helices α2, α3 and α10 and on the other by helices α4 – α7, which, taken together, constitute the canonical Rossmann fold centre. The other helices and strands are located around the fold centre. Unlike the strands constituting the β-sheet, strands β5 – β6 and β7 – β8 form two anti-parallel hairpin structures.


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)

Crystal structure of xoFabV.The structure consists of 13 α-helices and 11 β-strands, representing a classic Rossmann fold architecture. The secondary structures are shown in different colours and are labelled with the corresponding numbers. (A) Side view. (B) Top view. Figures 1–4 were made using PyMOL (DeLano Scientific, Palo Alto, California, USA; http://www.pymol.org).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026743-g001: Crystal structure of xoFabV.The structure consists of 13 α-helices and 11 β-strands, representing a classic Rossmann fold architecture. The secondary structures are shown in different colours and are labelled with the corresponding numbers. (A) Side view. (B) Top view. Figures 1–4 were made using PyMOL (DeLano Scientific, Palo Alto, California, USA; http://www.pymol.org).
Mentions: The SeMet-xoFabV crystal contains one monomer in the asymmetric unit. The density map was of high quality and allowed us to build an almost complete model containing 390 residues (C12-L401) but lacking the His tag and thrombin cleavage site at the N-terminus and A402. Each xoFabV monomer consists of 13 α-helices (α1 – α13) and 11 β-strands (β1 – β11) (Fig. 1). Strands β1 – β4 and β9 – 11 form a parallel β-sheet located at the centre of the structure. This β-sheet is flanked on one side by helices α2, α3 and α10 and on the other by helices α4 – α7, which, taken together, constitute the canonical Rossmann fold centre. The other helices and strands are located around the fold centre. Unlike the strands constituting the β-sheet, strands β5 – β6 and β7 – β8 form two anti-parallel hairpin structures.

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