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Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis.

Nanson JD, Forwood JK - PLoS ONE (2015)

Bottom Line: FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs.YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa.Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2678, Australia.

ABSTRACT
Ketoacyl-acyl carrier protein reductases (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI) has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG), the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

No MeSH data available.


Related in: MedlinePlus

The co-factor specificity and enzymatic activity of Yersinia pestis FabG (YpFabG).(A) The activity of YpFabG in the presence of NADH (•) or NADPH (□) confirmed YpFabG reductase activity is highly dependent upon NADPH as the co-factor. YpFabG was assayed in the presence of acetoacetyl-CoA and NADPH, by varying the concentration of either acetoacetyl-CoA (B) or NADPH (C) while maintaining a fixed concentration of the other. The Km values of acetoacetyl-CoA and NADPH were 329.9 ± 31.2 μM and 55.1 ± 3.8 μM respectively.
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pone.0141543.g004: The co-factor specificity and enzymatic activity of Yersinia pestis FabG (YpFabG).(A) The activity of YpFabG in the presence of NADH (•) or NADPH (□) confirmed YpFabG reductase activity is highly dependent upon NADPH as the co-factor. YpFabG was assayed in the presence of acetoacetyl-CoA and NADPH, by varying the concentration of either acetoacetyl-CoA (B) or NADPH (C) while maintaining a fixed concentration of the other. The Km values of acetoacetyl-CoA and NADPH were 329.9 ± 31.2 μM and 55.1 ± 3.8 μM respectively.

Mentions: The structures of YpFabG, particularly the presence of a charged pocket for the 2’ phosphate of NADPH, indicated the catalytic mechanism of YpFabG to be NADPH dependent. To confirm this, the ketoacyl reductase activity of YpFabG was assessed by the oxidation of NADH or NADPH in the presence of acetoacetyl-CoA, similarly to that described previously [7, 9, 13, 41, 42]. The enzymatic activity assays confirmed YpFabG reductase activity is highly dependent upon NADPH as the co-factor, with little reductase activity occurring in the presence of NADH (Fig 4). YpFabG was found to catalyse the reduction of acetoacetyl-CoA with a Km of 329.9 ± 31.2 μM in the presence of 0.9 mM NADPH. The Km for NADPH was 55.1 ± 3.8 μM in the presence of 1.5 mM acetoacetyl-CoA. Vmax with respect to acetoacetyl-CoA was 0.383 ± 0.010 μmoles min-1 μg-1.


Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis.

Nanson JD, Forwood JK - PLoS ONE (2015)

The co-factor specificity and enzymatic activity of Yersinia pestis FabG (YpFabG).(A) The activity of YpFabG in the presence of NADH (•) or NADPH (□) confirmed YpFabG reductase activity is highly dependent upon NADPH as the co-factor. YpFabG was assayed in the presence of acetoacetyl-CoA and NADPH, by varying the concentration of either acetoacetyl-CoA (B) or NADPH (C) while maintaining a fixed concentration of the other. The Km values of acetoacetyl-CoA and NADPH were 329.9 ± 31.2 μM and 55.1 ± 3.8 μM respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4635001&req=5

pone.0141543.g004: The co-factor specificity and enzymatic activity of Yersinia pestis FabG (YpFabG).(A) The activity of YpFabG in the presence of NADH (•) or NADPH (□) confirmed YpFabG reductase activity is highly dependent upon NADPH as the co-factor. YpFabG was assayed in the presence of acetoacetyl-CoA and NADPH, by varying the concentration of either acetoacetyl-CoA (B) or NADPH (C) while maintaining a fixed concentration of the other. The Km values of acetoacetyl-CoA and NADPH were 329.9 ± 31.2 μM and 55.1 ± 3.8 μM respectively.
Mentions: The structures of YpFabG, particularly the presence of a charged pocket for the 2’ phosphate of NADPH, indicated the catalytic mechanism of YpFabG to be NADPH dependent. To confirm this, the ketoacyl reductase activity of YpFabG was assessed by the oxidation of NADH or NADPH in the presence of acetoacetyl-CoA, similarly to that described previously [7, 9, 13, 41, 42]. The enzymatic activity assays confirmed YpFabG reductase activity is highly dependent upon NADPH as the co-factor, with little reductase activity occurring in the presence of NADH (Fig 4). YpFabG was found to catalyse the reduction of acetoacetyl-CoA with a Km of 329.9 ± 31.2 μM in the presence of 0.9 mM NADPH. The Km for NADPH was 55.1 ± 3.8 μM in the presence of 1.5 mM acetoacetyl-CoA. Vmax with respect to acetoacetyl-CoA was 0.383 ± 0.010 μmoles min-1 μg-1.

Bottom Line: FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs.YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa.Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2678, Australia.

ABSTRACT
Ketoacyl-acyl carrier protein reductases (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI) has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG), the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

No MeSH data available.


Related in: MedlinePlus