Slow onset inhibition of bacterial beta-ketoacyl-acyl carrier protein synthases by thiolactomycin

Abstract

Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the beta-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices alpha5 and alpha6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100-200 microg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton.

FIGURE 1.

The FAS-II pathway in M. tuberculosis and the structures of several natural product KAS inhibitors. In M. tuberculosis, the FAS-II pathway elongates the C18+ acyl-CoAs from the FAS-I pathway to C54-C56 fatty acids. The pathway is primed by the CoA-dependent β-ketoacyl-AcpM synthase FabH, which condenses the acyl-CoA with malonyl-AcpM to generate a β-ketoacyl-AcpM. The β-ketoacyl-AcpM is converted into a saturated enoyl-AcpM by the sequential actions of a β-ketoacyl-AcpM reductase (MabA), a dehydrase, and a trans-2-enoyl-AcpM reductase (InhA). Subsequent rounds of elongation are initiated by the KasA or KasB β-ketoacyl-AcpM synthases. KasA is thought to be responsible for the early rounds of fatty acid elongation. Also shown is the malonyl-CoA AcpM transacylase (FabD) responsible for the synthesis of malonyl-AcpM. β-Ketoacyl-AcpM synthase inhibitors include the natural products thiolactomycin, cerulenin, and platensimycin.

FIGURE 2.

Ping-pong catalytic mechanism for KasA. Acyl-enzyme formation occurs after nucleophilic attack of the active site cysteine (Cys-171 in KasA) on the carbonyl carbon of acyl-AcpM. This reaction is facilitated by the oxyanion hole formed by the amide groups of Cys-171 and Phe-404. Dissociation of AcpM and binding of the second substrate, malonyl-AcpM, is followed by decarboxylation and carbanion formation. Condensation and carbon-carbon bond formation occurs through a nucleophilic attack by the malonyl-AcpM carbanion on the acyl-KasA thioester carbonyl group to form the β-keto acyl-AcpM product and free enzyme. Decarboxylation of malonyl-AcpM and subsequent condensation with the acyl group are facilitated by two conserved histidines (His-311 and His-345 in KasA). In the mechanism shown a conserved phenylalanine is proposed to destabilize the malonate anion, thereby promoting decarboxylation, in line with previous proposals for the mechanism of KASIII enzymes as well as thiolase homologues such as chalcone synthase (21). We note that formation of the acetyl carbanion has also been proposed to occur by attack of water on the malonate group and elimination of bicarbonate (51); however, in the case of KasA, a conserved phenylalanine (Phe-237) is appropriately positioned to destabilize the malonate anion, and no structured water molecule can be observed in the x-ray structures of wild-type and mutant KasA (45).

SCHEME 1.

General scheme for the two-step formation of an enzyme-inhibitor complex. In slow onset inhibition, formation of the initial E-I complex is rapid compared with formation of E-I*.

FIGURE 3.

Interaction of the KASI enzymes with TLM. a, change in fluorescence when 1 μm apo-KasA is titrated by TLM is shown. The solid line is the best fit of the data to the Scatchard equation. b, fluorescence decay curves obtained after the addition of TLM to 1 μm C171Q KasA are shown. Data are shown for TLM concentrations 10, 15, 20, and 50 μm, and the solid lines are the best fit of the data to Equation 1. c, plot of k_obs as a function of TLM concentration obtained from the data shown in b, with the solid line showing the best fit to Equation 2. d, change in fluorescence when 1 μm apo-ecFabB is titrated by TLM is shown. The solid line is the best fit of the data to the Scatchard equation. e, fluorescence decay curves obtained after the addition of TLM to 1 μm acyl-ecFabB are shown. Data are shown for TLM concentrations 3, 5, 10, 20, and 40 μm, and the solid lines are the best fit of the data to Equation 1. f, shown is a plot of k_obs as a function of TLM concentration obtained from the data shown in e, with the solid line showing the best fit to Equation 2. In each case fluorescence emission was monitored at 337 nm.

FIGURE 4.

Interaction of the KASII enzymes with TLM. a, change in fluorescence is shown when 250 nm apo-FabF (closed circles) or 250 nm acyl-FabF (open circles) are titrated by TLM. In each case the black line is the best fit of the data to the Scatchard equation. b, change in fluorescence is shown when 1 μm apo-KasB (closed circles) or 1 μm acyl-KasB (open circles) are titrated by TLM. In each case the black line is the best fit of the data to the Scatchard equation.

SCHEME 2.

Scheme for the interaction of TLM with KasA.

FIGURE 5.

Interactions linking the KasA active site and helices α5 and α6. a, shown is the structure of wild-type and C171Q KasA bound to TLM. KasA is a homodimer and the wild-type subunits are colored cyan and magenta, whereas the C171Q subunits are colored yellow and silver. TLM is colored black. Arrows show the movement of helices α5 and α6 on adjacent subunits upon formation of the acyl-enzyme, mimicked in this case by mutation of the catalytic cysteine to a glutamine. Key residues that link the active site with helices α5 and α6 are shown as sticks. b, a detailed view shows the side chains, which alter their position upon transition from the free enzyme to the acyl-enzyme. Hydrogen-bond formation between the carbonyl oxygen of Gln-171 and the oxyanion hole NH donors Phe-204 and Gln-171 cause Phe-404 and loop 1 to move closer to loop 2 containing Met-277 and Val-278. The Met-277 side chain shifts closer to Met-146 at the base of helices α5 and α6 in the opposite subunit. This in turn decreases the distance between the Phe-147 residues from each subunit from 7.57 to 4.57 Å linking changes in one subunit with the corresponding structural changes in the second subunit. The figure was made using PyMOL (3) and the PDB entries 2WGD (wild type without TLM), 2WGE (wild type with TLM), 2WGF (C171Q without TLM), and 2WGG (C171Q with TLM).