Fig 1.
The fatty acid synthesis pathway of Yersinia pestis.
In contrast to the mammalian fatty acid synthesis pathway in which reactions are catalysed by a single protein (FAS or FASN), each acyltransferase, condensation, reduction, and dehydration reaction of the fatty acid synthesis pathway of Yersinia pestis is catalysed by a discrete enzyme (highlighted green). FabG (highlighted blue with red lettering) is a highly conserved and ubiquitously expressed enzyme, which performs the first of two reduction reactions within the pathway. Homologues from other organisms are not highlighted. Image adapted from the KEGG PATHWAY database [14, 15].
Table 1.
Data processing and model statistics.
Fig 2.
The tertiary structure of Yersinia pestis FabG (YpFabG).
(A) A cartoon representation of YpFabG TMAO showing the central seven strand twisted β-sheet flanked either side by α-helices. (B) A 2D depiction of the secondary structure features of YpFabG, with the two βαβαβ motifs and αααβ motif highlighted by grey squares. (C) The amino acid sequence of YpFabG, and corresponding secondary structure features. β-sheets are displayed yellow; α-helices are displayed red; loop regions are displayed green.
Fig 3.
Different Yersinia pestis FabG (YpFabG) crystal forms exhibit different loop conformations.
Despite similar crystallisation conditions, the structures of YpFabG TMAO and YpFabG NaBr exhibit differences within the loop regions surrounding the co-factor binding pocket. (A) Within our YpFabG NaBr structure (orange model), the region between Ser138 and Gly147 is highly disordered, with little visible electron density (red mesh), however this region is ordered in our YpFabG TMAO structure (cyan model; blue electron density map). (B) The helix-turn-helix motif of both YpFabG structures exhibit some disorder, however a greater portion of this motif is visible in the YpFabG TMAO structure. Electron density maps are 2Fo-Fc maps contoured to 1σ.
Fig 4.
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.
Fig 5.
The quaternary structure of Yersinia pestis FabG (YpFabG).
(A) A cartoon representation of the YpFabG TMAO homo-tetramer, the biological unit as indicated by PISA. (B) YpFabG forms a tetramer with two types of interfaces. The larger of the two interfaces in terms of buried surface area (A/D interface) is formed primarily through interactions between helices α4 and α5 of each monomer, which align antiparallel to form a four helix bundle. (C) The smaller of the two interfaces (A/B interface) is formed through interactions between helix α8, strand β7, and the adjoining loop region of each monomer, which align antiparallel, with the β7 strand of each monomer arranged to form a contiguous 14 strand β-sheet that spans the dimer interface. Opposing monomer secondary structure features are indicated by asterisks (*).
Table 2.
Hydrogen bonds and salt bridges of the YpFabG dimer interfaces.
Fig 6.
Comparison of bacterial FabG enzymes and the ketoreductase domain of the mammalian FAS.
(A) Superposition of FabG from Y. pestis (YpFabG TMAO; blue), E. coli (green), V. cholerae (cyan), and the ketoreductase (KR) domain of the mammalian FAS from S. scrofa (magenta) and H. sapiens (silver) reveals a highly conserved tertiary structure, however there are several differences surrounding the NADP(H) (yellow) binding site. (B) The mammalian FAS enzyme possesses two additional electropositive arginine residues, which appear to interact with the 3’ ribose phosphate of NADP(H) compared to bacterial FabG enzymes. (C) Additionally, the helix turn helix motif that caps the co-factor binding site appears shorter in the mammalian FAS compared to bacterial FabG enzymes. (D) Interestingly, the position of the active site lysine and the structurally conserved asparagine in the active site of the KR domain of the mammalian FAS appears switched in comparison to bacterial FabG enzymes, yet the position of the active site tyrosine is unchanged. Residues of H. sapiens and S. scrofa FAS are indicated by asterisks (*); active site residues common to both YpFabG and the mammalian FAS are not marked. (E) Sequence alignment of FabG from E. coli, V. cholerae, N. meningitidis, P. aeruginosa, 3α, 20β-hydroxysteroid dehydrogenase, and the KR domain of the mammalian FAS from H. sapiens and S. scrofa. Conserved residues are highlighted yellow; strictly conserved residues are highlighted red.
Fig 7.
Comparison of bacterial FabG and mammalian FAS ketoreductase domain inhibitor binding sites.
(A) The mammalian FAS ketoreductase (KR) domain (silver) inhibitor GSK2194069 (purple) binding site extends from the KR domain active site into the interface between the KR and pseudo-methyltransferase domains of the FAS complex [44]. In YpFabG (blue) and E. coli FabG (green) this site extends towards the A/B dimer interface at the centre of the tetramer, and appears incompatible with GSK2194069 binding due to clashes with residues Met143, Phe183, Met241, and Tyr242. (B) The allosteric inhibitors (orange) reported by Cukier et al. (2013) bind within the A/D dimer interfaces of FabG between helices α4 and α5 of each monomer, distorting the NADPH binding pocket to prevent co-factor binding [8]. Whilst the KR domain of mammalian FAS does not possess the same interfaces, superposition of bacterial FabG (cyan) and the mammalian FAS KR domains of H. sapiens (silver) and S. scrofa (magenta) reveals a region somewhat homologous to the A/D interface. However, two of the four helices of this site are truncated in comparison to FabG, thus such structural differences may be able to be exploited to reduce the affinity of the inhibitor for the mammalian FAS. Opposing monomer secondary structure features are indicated by asterisks (*).