Mechanism and Inhibition of the FabV Enoyl-ACP Reductase from Burkholderia mallei

Abstract

Enoyl-ACP reductases catalyze the final step in the elongation cycle of the bacterial fatty acid biosynthesis (FAS-II) pathway. Currently four distinct enoyl-ACP reductases have been identified, which are the products of the fabI, fabL, fabK and fabV genes. The FabV enoyl-ACP reductase is the most recent member of this enzyme class and was originally identified in Vibrio cholerae by Cronan and coworkers [Massengo-Tiasse and Cronan (2008) Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase, J. Biol. Chem. 283, 1308-1316. In the present work a detailed kinetic analysis of the mechanism of the FabV enzyme from Burkholderia mallei (bmFabV) has been undertaken, which reveals that bmFabV catalyzes a sequential Bi Bi mechanism with NADH binding first and NAD + dissociating last. The enzyme is a member of the short chain dehydrogenase/reductase superfamily in which the catalytic tyrosine (Y235) and lysine (K244) residues are organized in the consensus Tyr-(Xaa) 8 -Lys motif. The role of these active-site residues has been investigated using site-directed mutagenesis which has shown that both Y235 and K244 are involved in acid/base chemistry during substrate reduction. Sequence alignment and site-directed mutagenesis also identify a second lysine in the active site (K245) that has an important role in binding of the enoyl substrate. Due to interests in developing inhibitors of bmFabV, a detailed analysis of the inhibition of the enzyme by triclosan has been conducted showing that triclosan is a competitive inhibitor with respect to NADH and an uncompetitive inhibitor with respect to the substrate 2-dodecenoyl-CoA (K i = 0.4 µM). Combined with fluorescence binding experiments, it is concluded that triclosan binds to the enzyme-NAD + product complex which is in rapid and reversible equilibrium with other intermediates on the reaction pathway.The bacterial fatty acid biosynthesis (FAS-II) pathway is a validated yet relatively unexploited target for antimicrobial development (1-2). Fatty acids are important components of the cell membrane and are essential for bacterial viability (3-4). While eukaryotes synthesize fatty acids using a multi-domain enzyme complex (FAS-I), the FAS-II pathway found in prokaryotes consists of individual enzymes that catalyze each step of fatty chain elongation (5-7). Consequently, the fundamental structural differences that exist between the FAS-I and FAS-II pathways supports the proposal that compounds can be developed that selectively target bacterial fatty acid biosynthesis.The majority of the FAS-II enzymes are essential for bacterial viability (8) and detailed kinetic studies coupled with high resolution crystal structures have provided a solid foundation for the development of compounds that target this pathway (2,9). In particular, the enoyl-ACP reductase, which catalyzes the last reaction in each elongation circle, has been the most heavily targeted component of the pathway based on the discovery that antibacterial compounds such † This work was suppor...

Rational Design of Broad Spectrum Antibacterial Activity Based on a Clinically Relevant Enoyl-Acyl Carrier Protein (ACP) Reductase Inhibitor

“…Although the enol form of PT191 displays a similar putative binding geometry as PT179 (supplemental Fig. S1) and more closely resembles the potent diphenyl ether PT04 (31, 66), the increased desolvation costs (23,24,29,81). Residues that are likely responsible for the unique NADPH specificity, enhanced mobility (6), and enlarged binding pocket of saFabI are indicated by magenta, black, and red stars, respectively.…”

The Burkholderia pseudomallei Enoyl-Acyl Carrier Protein Reductase FabI1 Is Essential for In Vivo Growth and Is the Target of a Novel Chemotherapeutic with Efficacy

“…There are four known isoforms of enoyl-ACP reductases, FabI, FabV, FabK, and FabL (31)(32)(33)(34), and bioinformatics has identified that Burkholderia species carry genes that encode both FabI (BP1026B_I1187 and BP1026B_II0795) and FabV (BP1026B_ I1988) enoyl-ACP reductases. To begin to assess the possible role of each of the enoyl-ACP reductases, we performed enzymatic and in vitro and in vivo transcriptional analyses.…”

ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in <b><i>Escherichia coli</i></b>

“…The loss of interaction between residue 245 and the backbone could result in a loss of catalytic efficiency due to improper positioning of Y235. Mutation of that K245 to methionine in bmFabV has been shown to result in a 10-fold increase in K M [Lu and Tonge, 2010]. In contrast to other FabV homologs, ucFabV contains a glutamate instead of lysine at position 245.…”

“…These residues, corresponding to Y156 and K163 in FabI and Y235 and K244 in ucFabV, are likely necessary to stabilize the negatively charged reaction intermediate [Baldock et al, 1998]. Other groups have demonstrated that mutating the lysine to alanine within the YX n K motif in homologous enoyl reductases abolishes the ability of the protein to act as a reductase [Lu and Tonge, 2010;Parikh et al, 1999]. To test whether this residue is important for both triclosan resistance and reductase activity in ucFabV, we mutated lysine 244 to alanine and assayed for the ability of the mutant protein to confer reduced triclosan susceptibility on E. coli ( table 1 ).…”