Richard J. Heath

Mechanism of Triclosan Inhibition of Bacterial Fatty Acid Synthesis

Triclosan is a broad-spectrum antibacterial agent that inhibits bacterial fatty acid synthesis at the enoyl-acyl carrier protein reductase (FabI) step. Resistance to triclosan inEscherichia coli is acquired through a missense mutation in the fabI gene that leads to the expression of FabI[G93V]. The specific activity and substrate affinities of FabI[G93V] are similar to FabI. Two different binding assays establish that triclosan dramatically increases the affinity of FabI for NAD+. In contrast, triclosan does not increase the binding of NAD+to FabI[G93V]. The x-ray crystal structure of the FabI-NAD+-triclosan complex confirms that hydrogen bonds and hydrophobic interactions between triclosan and both the protein and the NAD+ cofactor contribute to the formation of a stable ternary complex, with the drug binding at the enoyl substrate site. These data show that the formation of a noncovalent “bi-substrate” complex accounts for the effectiveness of triclosan as a FabI inhibitor and illustrates that mutations in the FabI active site that interfere with the formation of a stable FabI-NAD+-triclosan ternary complex acquire resistance to the drug.

Broad Spectrum Antimicrobial Biocides Target the FabI Component of Fatty Acid Synthesis

The broad spectrum antibacterial properties of 2-hydroxydiphenyl ethers have been appreciated for decades, and their use in consumer products is rapidly increasing. We identify the enoyl-acyl carrier protein reductase (fabI) component of the type II fatty acid synthase system as the specific cellular target for these antibacterials. Biologically active 2-hydroxydiphenyl ethers effectively inhibit fatty acid synthesis in vivo and FabI activity in vitro. Resistant mechanisms include up-regulation of fabI expression and spontaneously arising missense mutations in the fabI gene. These results contradict the view that these compounds directly disrupt membranes and suggest that their widespread use will select for resistant bacterial populations.

Enoyl-acyl carrier protein-reductase (fabI) plays a determinant role in completing cycles of fatty acid elongation in Escherichia coli

The role of enoyl-acyl carrier protein (ACP) reductase (E.C. 1.3.1.9), the product of the fabI gene, was investigated in the type II, dissociated, fatty acid synthase system of Escherichia coli. All of the proteins required to catalyze one cycle of fatty acid synthesis from acetyl-CoA plus malonyl-CoA to butyryl-ACP in vitro were purified. These proteins were malonyl-CoA:ACP transacylase (fabD), β-ketoacyl-ACP synthase III (fabH), β-ketoacyl-ACP reductase (fabG), β-hydroxydecanoyl-ACP dehydrase (fabA), and enoyl-ACP reductase (fabI). Unlike the other enzymes in the cycle, FabA did not efficiently convert its substrate β-hydroxybutyryl-ACP to crotonyl-ACP, but rather the equilibrium favored formation of β-hydroxybutyryl-ACP over crotonyl-ACP by a ratio of 9:1. The amount of butyryl-ACP formed depended on the amount of FabI protein added to the assay. Extracts from fabI(Ts) mutants accumulated β-hydroxybutyryl-ACP, and the addition of FabI protein to the fabI(Ts) extract restored both butyryl-ACP and long-chain acyl-ACP synthesis. FabI was verified to be the only enoyl-ACP reductase required for the synthesis of fatty acids by demonstrating that purified FabI was required for the elongation of both long-chain saturated and unsaturated fatty acids. These results were corroborated by analysis of the intracellular ACP pool composition in fabI(Ts) mutants that showed β-hydroxybutyryl-ACP and crotonyl-ACP accumulated at the nonpermissive temperature in the same ratio found in the fabI(Ts) extracts and in the in vitro reconstruction experiments that lacked FabI. We conclude that FabI is the only enoyl-ACP reductase involved in fatty acid synthesis in E. coli and that the activity of this enzyme plays a determinant role in completing cycles of fatty acid biosynthesis.

Lipid biosynthesis as a target for antibacterial agents.

Fatty acid biosynthesis, the first stage in membrane lipid biogenesis, is catalyzed in most bacteria by a series of small, soluble proteins that are each encoded by a discrete gene (Fig. 1; Table 1). This arrangement is termed the type II fatty acid synthase (FAS) system and contrasts sharply with the type I FAS of eukaryotes which is a dimer of a single large, multifunctional polypeptide. Thus, the bacterial pathway offers several unique sites for selective inhibition by chemotherapeutic agents. The site of action of isoniazid, used in the treatment of tuberculosis for 50 years, and the consumer antimicrobial agent triclosan were revealed recently to be the enoyl-ACP reductase of the type II FAS. The fungal metabolites, cerulenin and thiolactomycin, target the condensing enzymes of the bacterial pathway while the dehydratase/isomerase is inhibited by a synthetic acetylenic substrate analogue. Transfer of fatty acids to the membrane has also been inhibited via interference with the first acyltransferase step, while a new class of drugs targets lipid A synthesis. This review will summarize the data generated on these inhibitors to date, and examine where additional efforts will be required to develop new chemotherapeutics to help combat microbial infections.

Insulin antagonizes ischemia-induced Thr172 phosphorylation of AMP-activated protein kinase alpha-subunits in heart via hierarchical phosphorylation of Ser485/491.

Previous studies showed that insulin antagonizes AMP-activated protein kinase activation by ischemia and that protein kinase B might be implicated. Here we investigated whether the direct phosphorylation of AMP-activated protein kinase by protein kinase B might participate in this effect. Protein kinase B phosphorylated recombinant bacterially expressed AMP-activated protein kinase heterotrimers at Ser485 of the α1-subunits. In perfused rat hearts, phosphorylation of the α1/α2 AMP-activated protein kinase subunits on Ser485/Ser491 was increased by insulin and insulin pretreatment decreased the phosphorylation of the α-subunits at Thr172 in a subsequent ischemic episode. It is proposed that the effect of insulin to antagonize AMP-activated protein kinase activation involves a hierarchical mechanism whereby Ser485/Ser491 phosphorylation by protein kinase B reduces subsequent phosphorylation of Thr172 by LKB1 and the resulting activation of AMP-activated protein kinase.

Defining the Mechanism of Activation of AMP-activated Protein Kinase by the Small Molecule A-769662, a Member of the Thienopyridone Family

AMP-activated protein kinase (AMPK) plays a key role in maintaining energy homeostasis. Activation of AMPK in peripheral tissues has been shown to alleviate the symptoms of metabolic diseases, such as type 2 diabetes, and consequently AMPK is a target for treatment of these diseases. Recently, a small molecule activator (A-769662) of AMPK was identified that had beneficial effects on metabolism in ob/ob mice. Here we show that A-769662 activates AMPK both allosterically and by inhibiting dephosphorylation of AMPK on Thr-172, similar to the effects of AMP. A-769662 activates AMPK harboring a mutation in the γ subunit that abolishes activation by AMP. An AMPK complex lacking the glycogen binding domain of the β subunit abolishes the allosteric effect of A-769662 but not the allosteric activation by AMP. Moreover, mutation of serine 108 to alanine, an autophosphorylation site within the glycogen binding domain of the β1 subunit, almost completely abolishes activation of AMPK by A-769662 in cells and in vitro, while only partially reducing activation by AMP. Based on our results we propose a model for activation of AMPK by A-769662. Importantly, this model may provide clues for understanding the mechanism by which AMP leads to activation of AMPK, which in turn may help in the identification of other AMPK activators.

beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis.

A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes. We examined the biochemical basis for the production of branched-chain fatty acids by gram-positive bacteria. Two genes that were predicted to encode homologs of the beta-ketoacyl-acyl carrier protein synthase III of Escherichia coli (eFabH) were identified in the Bacillus subtilis genome. Their protein products were expressed, purified, and biochemically characterized. Both B. subtilis FabH homologs, bFabH1 and bFabH2, carried out the initial condensation reaction of fatty acid biosynthesis with acetyl-coenzyme A (acetyl-CoA) as a primer, although they possessed lower specific activities than eFabH. bFabH1 and bFabH2 also utilized iso- and anteiso-branched-chain acyl-CoA primers as substrates. eFabH was not able to accept these CoA thioesters. Reconstitution of a complete round of fatty acid synthesis in vitro with purified E. coli proteins showed that eFabH was the only E. coli enzyme incapable of using branched-chain substrates. Expression of either bFabH1 or bFabH2 in E. coli resulted in the appearance of a branched-chain 17-carbon fatty acid. Thus, the substrate specificity of FabH is an important determinant of branched-chain fatty acid production.

The 1.8 Å crystal structure and active-site architecture of β-ketoacyl-acyl carrier protein synthase III (FabH) from Escherichia coli

BACKGROUND beta-Ketoacyl-acyl carrier protein synthase III (FabH) initiates elongation in type II fatty acid synthase systems found in bacteria and plants. FabH is a ubiquitous component of the type II system and is positioned ideally in the pathway to control the production of fatty acids. The elucidation of the structure of FabH is important for the understanding of its regulation by feedback inhibition and its interaction with drugs. Although the structures of two related condensing enzymes are known, the roles of the active-site residues have not been experimentally tested. RESULTS The 1.8 A crystal structure of FabH was determined using a 12-site selenium multiwavelength anomalous dispersion experiment. The active site (Cys112, His244 and Asn274) is formed by the convergence of two alpha helices and is accessed via a narrow hydrophobic tunnel. Hydrogen-bonding networks that include two tightly bound water molecules fix the positions of His244 and Asn274, which are critical for the decarboxylation and condensation reactions. Surprisingly, the His244-->Ala mutation does not affect the transacylation reaction suggesting that His244 has only a minor influence on the nucleophilicity of Cys112. CONCLUSIONS The histidine and asparagine active-site residues are both required for the decarboxylation step in the condensation reaction. The nucleophilicity of the active-site cysteine is enhanced by the alpha-helix dipole effect, and an oxyanion hole promotes the formation of the tetrahedral transition state.

Inhibitors of fatty acid synthesis as antimicrobial chemotherapeutics

Abstract. Fatty acid biosynthesis is an emerging target for the development of novel antibacterial chemotherapeutics. The dissociated bacterial system is substantially different from the large, multifunctional protein of mammals, and many possibilities exist for type-selective drugs. Several compounds, both synthetic and natural, target bacterial fatty acid synthesis. Three compounds target the FabI enoyl-ACP reductase step; isoniazid, a clinically used antituberculosis drug, triclosan, a widely used consumer antimicrobial, and diazaborines. In addition, cerulenin and thiolactomycin, two fungal products, inhibit the FabH, FabB and FabF condensation enzymes. Finally, the synthetic reaction intermediates BP1 and decynoyl-N-acetyl cysteamine inhibit the acetyl-CoA carboxylase and dehydratase isomerase steps, respectively. The mechanisms of action of these compounds, as well as the potential development of new drugs targeted against this pathway, are discussed.

Microbiology: A triclosan-resistant bacterial enzyme

Triclosan is an antimicrobial agent that is widely used in a variety of consumer products and acts by inhibiting one of the highly conserved enzymes (enoyl-ACP reductase, or FabI) of bacterial fatty-acid biosynthesis. But several key pathogenic bacteria do not possess FabI, and here we describe a unique triclosan-resistant flavoprotein, FabK, that can also catalyse this reaction in Streptococcus pneumoniae. Our finding has implications for the development of FabI-specific inhibitors as antibacterial agents.