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. At present, 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 co-workers [Massengo-Tiasse, R. P., and Cronan, J. E. (2008) Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase. J. Biol. Chem. 283, 1308-1316]. In this 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. Because of 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 muM). In combination with fluorescence binding experiments, we conclude that triclosan binds to the enzyme-NAD(+) product complex which is in rapid and reversible equilibrium with other intermediates on the reaction pathway.

FIGURE 1

Two-substrate steady-state kinetics. Initial velocity patterns: (A) 1/v versus 1/[NADH] double-reciprocal plots in which the DD-CoA concentration was fixed at 6 (□), 12 (Δ) and 18 µM (○) and (B) 1/v versus 1/[DD-CoA] double-reciprocal plots in which the NADH concentration was fixed at 33 (□), 110 (Δ) and 250 µM (○).

FIGURE 1

Two-substrate steady-state kinetics. Initial velocity patterns: (A) 1/v versus 1/[NADH] double-reciprocal plots in which the DD-CoA concentration was fixed at 6 (□), 12 (Δ) and 18 µM (○) and (B) 1/v versus 1/[DD-CoA] double-reciprocal plots in which the NADH concentration was fixed at 33 (□), 110 (Δ) and 250 µM (○).

FIGURE 2

Product inhibition studies to determine the substrate binding order. Assays were performed by varying the concentration of one substrate at a fixed concentration of the second substrate and in the presence of one of the products of the reaction. (A) NADH varied in the presence of NAD⁺ (0 (□), 55 (○) and 110 µM (Δ)) with DD-CoA fixed at 35 µM. (B) DD-CoA varied in the presence of NAD⁺ (0 (○), 55 (Δ) and 110 µM (□)) with NADH fixed at 250 µM. (C) NADH varied in the presence of lauryl-CoA (0 (○), 50 (□) and 100 µM (Δ)) with DD-CoA fixed at 35 µM. (D) DD-CoA varied in the presence of lauryl-CoA (0 (○), 50 (□) and 100 µM (Δ)) with NADH fixed at 250 µM.

FIGURE 2

Product inhibition studies to determine the substrate binding order. Assays were performed by varying the concentration of one substrate at a fixed concentration of the second substrate and in the presence of one of the products of the reaction. (A) NADH varied in the presence of NAD⁺ (0 (□), 55 (○) and 110 µM (Δ)) with DD-CoA fixed at 35 µM. (B) DD-CoA varied in the presence of NAD⁺ (0 (○), 55 (Δ) and 110 µM (□)) with NADH fixed at 250 µM. (C) NADH varied in the presence of lauryl-CoA (0 (○), 50 (□) and 100 µM (Δ)) with DD-CoA fixed at 35 µM. (D) DD-CoA varied in the presence of lauryl-CoA (0 (○), 50 (□) and 100 µM (Δ)) with NADH fixed at 250 µM.

FIGURE 2

Product inhibition studies to determine the substrate binding order. Assays were performed by varying the concentration of one substrate at a fixed concentration of the second substrate and in the presence of one of the products of the reaction. (A) NADH varied in the presence of NAD⁺ (0 (□), 55 (○) and 110 µM (Δ)) with DD-CoA fixed at 35 µM. (B) DD-CoA varied in the presence of NAD⁺ (0 (○), 55 (Δ) and 110 µM (□)) with NADH fixed at 250 µM. (C) NADH varied in the presence of lauryl-CoA (0 (○), 50 (□) and 100 µM (Δ)) with DD-CoA fixed at 35 µM. (D) DD-CoA varied in the presence of lauryl-CoA (0 (○), 50 (□) and 100 µM (Δ)) with NADH fixed at 250 µM.

FIGURE 2

Product inhibition studies to determine the substrate binding order. Assays were performed by varying the concentration of one substrate at a fixed concentration of the second substrate and in the presence of one of the products of the reaction. (A) NADH varied in the presence of NAD⁺ (0 (□), 55 (○) and 110 µM (Δ)) with DD-CoA fixed at 35 µM. (B) DD-CoA varied in the presence of NAD⁺ (0 (○), 55 (Δ) and 110 µM (□)) with NADH fixed at 250 µM. (C) NADH varied in the presence of lauryl-CoA (0 (○), 50 (□) and 100 µM (Δ)) with DD-CoA fixed at 35 µM. (D) DD-CoA varied in the presence of lauryl-CoA (0 (○), 50 (□) and 100 µM (Δ)) with NADH fixed at 250 µM.

FIGURE 3

Sequence alignment of active site residues of the enoyl-ACP reductases from Escherichia coli, Bacillus subtilis, Burkholderia mallei, Yesinia pestis, Clostridium acetobutylicum, Pseudomonas aeruginosa, Variovorax paradoxus, Methylobacillus flagellatus, Erwinia tasmaniensis and Vibrio cholerae. Conserved residues are labeled with an asterix. The sequence alignment was performed using Clustal W (48), and the figure was made using Jalview (49).

FIGURE 4

Proposed hydrogen bonding network between the three active site residues (Y235, K244 and K245), cofactor and the enoyl–ACP substrate. Y235 stabilizes the transition state of the enoyl-ACP substrate through a hydrogen bond with the carbonyl group while K244 interacts directly with Y235 as proposed in the SDR dehydrogenases. K244 is also shown interacting with the NADH ribose hydroxyl and the secondary hydroxyl group in the ACP pantetheine, in order to account for the role of this residue in binding cofactor and substrate. K245 is shown interacting with the thioester carbonyl group, to account for the effect of mutating this residue on k_cat, and also with the ACP pantetheine.

FIGURE 5

Progress curve analysis of ftuFabI and bmFabV. Progress curves for ftuFabI (2 nM) in the presence of DD-CoA (200 µM), NADH (250 µM) and either 0 (●) or 60 nM (▲) triclosan. Progress curves for bmFabV (2 nM) in the presence of DD-CoA (200 µM), NADH (250 µM) and either 0 (○) or 2 µM (∆) triclosan.

FIGURE 6

Mechanism of bmFabV inhibition by triclosan. Assays were performed by varying the concentration of one substrate at a fixed concentration of the second substrate constant and at various concentrations of triclosan. (A) NADH varied in the presence of triclosan (0 (○), 0.8 (∆) and 1.6 µM (□)) with DD-CoA fixed at 35 µM. (B) DD-CoA varied in the presence of triclosan (0 (○), 0.8 (◊) and 1.6 µM (∆)) with NADH fixed at 250 µM.