Kinetic determinants of the interaction of enoyl-ACP reductase from Plasmodium falciparum with its substrates and inhibitors.

We have recently demonstrated that Plasmodium falciparum, unlike its human host, has the type II fatty acid synthase, in which steps of fatty acid biosynthesis are catalyzed by independent enzymes. This difference could be successfully exploited in the design of drugs specifically targeted at the different enzymes of this pathway in P. falciparum, without affecting the corresponding enzymes in humans. The importance of enoyl-ACP reductase (FabI) in the fatty acid biosynthesis pathway makes it an important target in antimalarial therapy. We report here the initial characterization of Plasmodium FabI expressed in Escherichia coli. The K(m) values of the enzyme for crotonyl-CoA and NADH were derived as 165 and 33 microM, respectively. Triclosan shows competitive kinetics with respect to NADH but is uncompetitive with respect to NAD(+), which shows that the binding of triclosan to the enzyme is facilitated in the presence of NAD(+).     

Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase

Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia coli that is the target of the antibacterial compound, triclosan. However, some Gram-positive bacteria are naturally resistant to triclosan due to the presence of the triclosan-resistant enoyl-ACP reductase isoforms, FabK and FabL. The genome of the Gram-negative bacterium, Vibrio cholerae lacks a gene encoding a homologue of any of the three known enoyl-ACP reductase isozymes suggesting that this organism encodes a novel fourth enoyl-ACP reductase isoform. We report that this is the case. The gene encoding the new isoform, called FabV, was isolated by complementation of a conditionally lethal E. coli fabI mutant strain and was shown to restore fatty acid synthesis to the mutant strain both in vivo and in vitro. Like FabI and FabL, FabV is a member of the short chain dehydrogenase reductase superfamily, although it is considerably larger (402 residues) than either FabI (262 residues) or FabL (250 residues). The FabV, FabI and FabL sequences can be aligned, but only poorly. Alignment requires many gaps and yields only 15% identical residues. Thus, FabV defines a new class of enoyl-ACP reductase. The native FabV protein has been purified to homogeneity and is active with both crotonyl-ACP and the model substrate, crotonyl-CoA. In contrast to FabI and FabL, FabV shows a very strong preference for NADH over NADPH. Expression of FabV in E. coli results in markedly increased resistance to triclosan and the purified enzyme is much more resistant to triclosan than is E. coli FabI.     

Triclosan offers protection against blood stages of malaria by inhibiting enoyl-ACP reductase of Plasmodium falciparum

The antimicrobial biocide triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol] potently inhibits the growth of Plasmodium falciparum in vitro and, in a mouse model, Plasmodium berghei in vivo. Inhibition of [14C]acetate and [14C]malonyl-CoA incorporation into fatty acids in vivo and in vitro, respectively, by triclosan implicate FabI as its target. Here we demonstrate that the enoyl-ACP reductase purified from P. falciparum is triclosan sensitive. Also, we present the evidence for the existence of FabI gene in P. falciparum. We establish the existence of the de novo fatty acid biosynthetic pathway in this parasite, and identify a key enzyme of this pathway for the development of new antimalarials.     

The enoyl-[acyl-carrier-protein] reductases FabI and FabL from Bacillus subtilis

Enoyl-[acyl-carrier-protein] (ACP) reductase is a key enzyme in type II fatty-acid synthases that catalyzes the last step in each elongation cycle. The FabI component of Bacillus subtilis (bsFabI) was identified in the genomic data base by homology to the Escherichia coli protein. bsFabI was cloned and purified and exhibited properties similar to those of E. coli FabI, including a marked preference for NADH over NADPH as a cofactor. Overexpression of the B. subtilis fabI gene complemented the temperature-sensitive growth phenotype of an E. coli fabI mutant. Triclosan was a slow-binding inhibitor of bsFabI and formed a stable bsFabI.NAD(+). triclosan ternary complex. Analysis of the B. subtilis genomic data base revealed a second open reading frame (ygaA) that was predicted to encode a protein with a relatively low overall similarity to FabI, but contained the Tyr-Xaa(6)-Lys enoyl-ACP reductase catalytic architecture. The purified YgaA protein catalyzed the NADPH-dependent reduction of trans-2-enoyl thioesters of both N-acetylcysteamine and ACP. YgaA was reversibly inhibited by triclosan, but did not form the stable ternary complex characteristic of the FabI proteins. Expression of YgaA complemented the fabI(ts) defect in E. coli and conferred complete triclosan resistance. Single knockouts of the ygaA or fabI gene in B. subtilis were viable, but double knockouts were not obtained. The fabI knockout was as sensitive as the wild-type strain to triclosan, whereas the ygaA knockout was 250-fold more sensitive to the drug. YgaA was renamed FabL to denote the discovery of a new family of proteins that carry out the enoyl-ACP reductase step in type II fatty-acid synthases.     

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.     

Crystallographic analysis of triclosan bound to enoyl reductase

Molecular genetic studies with strains of Escherichia coli resistant to triclosan, an ingredient of many anti-bacterial household goods, have suggested that this compound works by acting as an inhibitor of enoyl reductase (ENR) and thereby blocking lipid biosynthesis. We present structural analyses correlated with inhibition data, on the complexes of E. coli and Brassica napus ENR with triclosan and NAD(+) which reveal how triclosan acts as a site-directed, picomolar inhibitor of the enzyme by mimicking its natural substrate. Elements of both the protein and the nucleotide cofactor play important roles in triclosan recognition, providing an explanation for the factors controlling its tight binding to the enzyme and for the emergence of triclosan resistance.     

Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase.

The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P. falciparum enoyl acyl carrier protein reductase gene, which encodes a 50-kDa protein (PfENR) predicted to target to the unique parasite apicoplast. Purified PfENR was crystallized, and its structure resolved as a binary complex with NADH, a ternary complex with triclosan and NAD(+), and as ternary complexes bound to the triclosan analogs 1 and 2 with NADH. Novel structural features were identified in the PfENR binding loop region that most closely resembled bacterial homologs; elsewhere the protein was similar to ENR from the plant Brassica napus (root mean square for Calphas, 0.30 A). Triclosan and its analogs 1 and 2 killed multidrug-resistant strains of intra-erythrocytic P. falciparum parasites at sub to low micromolar concentrations in vitro. These data define the structural basis of triclosan binding to PfENR and will facilitate structure-based optimization of PfENR inhibitors.     

A study of the structure-activity relationship for diazaborine inhibition of Escherichia coli enoyl-ACP reductase

Enoyl acyl carrier protein (ACP) reductase catalyses the last reductive step of fatty acid biosynthesis, reducing the enoyl group of a growing fatty acid chain attached to ACP to its acyl product using NAD(P)H as the cofactor. This enzyme is the target for the diazaborine class of antibacterial agents, the biocide triclosan, and one of the targets for the front-line anti-tuberculosis drug isoniazid. The structures of complexes of Escherichia coli enoyl-ACP reductase (ENR) from crystals grown in the presence of NAD+ and a family of diazaborine compounds have been determined. Analysis of the structures has revealed that a mobile loop in the structure of the binary complex with NAD+ becomes ordered on binding diazaborine/NAD+ but displays a different conformation in the two subunits of the asymmetric unit. The work presented here reveals how, for one of the ordered conformations adopted by the mobile loop, the mode of diazaborine binding correlates well with the activity profiles of the diazaborine family. Additionally, diazaborine binding provides insights into the pocket on the enzyme surface occupied by the growing fatty acid chain.     

Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site

Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements. The effects of ligands of dihydrofolate reductase on the reaction of E. coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase. The E. coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim. Methotrexate was studied in detail and proved to bind to an ATP binding site of the E. coli B enzyme with K1 value of 0.1 mM. The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase. These analyses would indicate that the homologous portion of the amino acid sequence of the E. coli B enzyme provides the ATP binding site. This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.     

Inhibitor binding studies on enoyl reductase reveal conformational changes related to substrate recognition.

Enoyl acyl carrier protein reductase (ENR) is involved in fatty acid biosynthesis. In Escherichia coli this enzyme is the target for the experimental family of antibacterial agents, the diazaborines, and for triclosan, a broad spectrum antimicrobial agent. Biochemical studies have suggested that the mechanism of diazaborine inhibition is dependent on NAD(+) and not NADH, and resistance of Brassica napus ENR to diazaborines is thought to be due to the replacement of a glycine in the active site of the E. coli enzyme by an alanine at position 138 in the plant homologue. We present here an x-ray analysis of crystals of B. napus ENR A138G grown in the presence of either NAD(+) or NADH and the structures of the corresponding ternary complexes with thienodiazaborine obtained either by soaking the drug into the crystals or by co-crystallization of the mutant with NAD(+) and diazaborine. Analysis of the ENR A138G complex with diazaborine and NAD(+) shows that the site of diazaborine binding is remarkably close to that reported for E. coli ENR. However, the structure of the ternary ENR A138G-NAD(+)-diazaborine complex obtained using co-crystallization reveals a previously unobserved conformational change affecting 11 residues that flank the active site and move closer to the nicotinamide moiety making extensive van der Waals contacts with diazaborine. Considerations of the mode of substrate binding suggest that this conformational change may reflect a structure of ENR that is important in catalysis.     

Elucidation of sulfadoxine resistance with structural models of the bifunctional Plasmodium falciparum dihydropterin pyrophosphokinase-dihydropteroate synthase

Resistance of the most virulent human malaria parasite, Plasmodium falciparum, to antifolates is spreading with increasing speed, especially in Africa. Antifolate resistance is mainly caused by point mutations in the P. falciparum dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) target proteins. Homology models of the bifunctional P. falciparum dihydropterin pyrophosphokinase-dihydropteroate synthase (PPPK-DHPS) enzyme as well as the separate domains complete with bound substrates were constructed using the crystal structures of Saccharomyces cerevisiae (PPPK-DHPS), Mycobacterium tuberculosis (DHPS), Bacillus anthracis (DHPS), and Escherichia coli (PPPK) as templates. The resulting structures were subsequently solvated and refined using molecular dynamics. The active site residues of DHPS are highly conserved in S. cerevisiae, M. tuberculosis, E. coli, S. aureus, and B. anthracis, an attribute also shared by P. falciparum DHPS. Sulfadoxine was superimposed into the equivalent position of the p-aminobenzoic acid substrate and its binding parameters were refined using minimization and molecular dynamics. Sulfadoxine appears to interact mainly with P. falciparum DHPS mainly through hydrophobic interactions. Rational explanations are provided by the model for the sulfadoxine resistance-causing effects of four of the five known mutations in P. falciparum DHPS. A possible structure for the bifunctional PPPK-DHPS was derived from the structure from the S. cerevisiae bifunctional enzyme. The active site residues of P. falciparum PPPK are also conserved when compared to S. cerevisiae, Haemophilus influenzae, and E. coli. The informative nature of these models opens up avenues for structure-based drug design approaches toward the development of alternative and more effective inhibitors of P. falciparum PPPK-DHPS.     

Structure-activity studies of the inhibition of FabI,the enoyl reductase from Escherichia coli,by triclosan: kinetic analysis of mutant FabIs

Triclosan, a common antibacterial additive used in consumer products, is an inhibitor of FabI, the enoyl reductase enzyme from type II bacterial fatty acid biosynthesis. In agreement with previous studies [Ward, W. H., Holdgate, G. A., Rowsell, S., McLean, E. G., Pauptit, R. A., Clayton, E., Nichols, W. W., Colls, J. G., Minshull, C. A., Jude, D. A., Mistry, A., Timms, D., Camble, R., Hales, N. J., Britton, C. J., and Taylor, I. W. (1999) Biochemistry 38, 12514-12525], we report here that triclosan is a slow, reversible, tight binding inhibitor of the FabI from Escherichia coli. Triclosan binds preferentially to the E.NAD(+) form of the wild-type enzyme with a K(1) value of 23 pM. In agreement with genetic selection experiments [McMurry, L. M., Oethinger, M., and Levy, S. B. (1998) Nature 394, 531-532], the affinity of triclosan for the FabI mutants G93V, M159T, and F203L is substantially reduced, binding preferentially to the E.NAD(+) forms of G93V, M159T, and F203L with K(1) values of 0.2 microM, 4 nM, and 0.9 nM, respectively. Triclosan binding to the E.NADH form of F203L can also be detected and is defined by a K(2) value of 51 nM. We have also characterized the Y156F and A197M mutants to compare and contrast the binding of triclosan to InhA, the homologous enoyl reductase from Mycobacterium tuberculosis. As observed for InhA, Y156F FabI has a decreased affinity for triclosan and the inhibitor binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3 and 30 nM, respectively. The replacement of A197 with Met has no impact on triclosan affinity, indicating that differences in the sequence of the conserved active site loop cannot explain the 10000-fold difference in affinities of FabI and InhA for triclosan.     

Molecular genetic analysis of enoyl-acyl carrier protein reductase inhibition by diazaborine

Diazaborine and isoniazid are, at first sight, unrelated anti-bacterial agents that inhibit the enoyl-ACP reductase (ENR) of Escherichia coli and Mycobacterium tuberculosis respectively. The crystal structures of these enzymes including that of the diazaborine-inhibited E. coli ENR have been obtained at high resolution. Site-directed mutagenesis was used to study the importance of amino acid residues in diazaborine susceptibility and enzyme function. The results show that drug binding and inhibition require the presence of a glycine residue at position 93 of E. coli ENR or at the structurally equivalent position in the plant homologue, which is naturally resistant to the drug. The data confirm the hypothesis that any amino acid side-chain other than hydrogen at this position within the three-dimensional structure of these enzymes will affect diazaborine resistance by encroaching into the drug binding site. Substitutions of Gly-93 by amino acids with small side-chains, such as serine, alanine, cysteine and valine, hardly affected the catalytic parameters and rendered the bacterial host resistant to the drug. Larger amino acid side-chains, such as that of arginine, histidine, lysine and glutamine, completely inactivated the activity of the enzyme.     

Enoyl-ACP reductase (FabI) of Haemophilus influenzae: steady-state kinetic mechanism and inhibition by triclosan and hexachlorophene

Steady-state kinetics, equilibrium binding, and primary substrate kinetic isotope effect studies revealed that the reduction of crotonyl-CoA by NADH, catalyzed by Haemophilus influenzae enoyl-ACP reductase (FabI), follows a rapid equilibrium random kinetic mechanism with negative interaction among the substrates. Two biphenyl inhibitors, triclosan and hexachlorophene, were studied in the context of the kinetic mechanism. IC(50) values for triclosan in the presence and absence of NAD(+) were 0.1 +/- 0.02 and 2.4 +/- 0.02 microM, respectively, confirming previous observations that the E-NAD(+) complex binds triclosan more tightly than the free enzyme. Preincubation of the enzyme with triclosan and NADH suggested that the E-NADH complex is the active triclosan binding species as well. These results were reinforced by measurement of binding kinetic transients. Intrinsic protein fluorescence changes induced by binding of 20 microM triclosan to E, E-NADH, E-NAD(+), and E-crotonyl-CoA occur at rates of 0.0124 +/- 0.001, 0.0663 +/- 0.002, 0.412 +/- 0.01, and 0.0069 +/- 0.0001 s(-1), respectively. The rate of binding decreased with increasing crotonyl-CoA concentrations in the E-crotonyl-CoA complex, and the extrapolated rate at zero concentration of crotonyl-CoA corresponded to the rate observed for the binding to the free enzyme. This suggests that triclosan and the acyl substrate share a common binding site. Hexachlorophene inhibition, on the other hand, was NAD(+)- and time-independent; and the calculated IC(50) value was 2.5 +/- 0.4 microM. Steady-state inhibition patterns did not allow the mode of inhibition to be unambiguously determined, but binding kinetics suggested that free enzyme, E-NAD(+), and E-crotonyl-CoA have similar affinity for hexachlorophene, since the k(obs)s were in the same range of 20-24 s(-1). When the E-NADH complex was mixed with hexachlorophene ligand, concentration-independent fluorescence quenching at 480 nm was observed, suggesting at least partial competition between NADH and hexachlorophene for the same binding site. Mutual exclusivity studies, together with the above-discussed results, indicate that triclosan and hexachlorophene bind at different sites of H. influenzae FabI.     

Targeting tuberculosis and malaria through inhibition of Enoyl reductase: compound activity and structural data

Tuberculosis and malaria together result in an estimated 5 million deaths annually. The spread of multidrug resistance in the most pathogenic causative agents, Mycobacterium tuberculosis and Plasmodium falciparum, underscores the need to identify active compounds with novel inhibitory properties. Although genetically unrelated, both organisms use a type II fatty-acid synthase system. Enoyl acyl carrier protein reductase (ENR), a key type II enzyme, has been repeatedly validated as an effective antimicrobial target. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the M. tuberculosis and P. falciparum enzyme (referred to as InhA and PfENR, respectively). The crystal structure of InhA complexed with NAD+ and one of the inhibitors was determined to elucidate the mode of binding. Structural analysis of InhA with the broad spectrum antimicrobial triclosan revealed a unique stoichiometry where the enzyme contained either a single triclosan molecule, in a configuration typical of other bacterial ENR:triclosan structures, or harbored two triclosan molecules bound to the active site. Significantly, these compounds do not require activation and are effective against wild-type and drug-resistant strains of M. tuberculosis and P. falciparum. Moreover, they provide broader chemical diversity and elucidate key elements of inhibitor binding to InhA for subsequent chemical optimization.     

Structural basis for triclosan and NAD binding to enoyl-ACP reductase of Plasmodium falciparum

Recent discovery of type II fatty acid synthase in the malarial parasite Plasmodium falciparum responsible for the most debilitating form of the disease in humans makes it ideal as a target for the development of novel antimalarials. Also, the identification of the enoyl-acyl carrier protein reductase from P. falciparum and the demonstration of its inhibition by triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol], a potent antibacterial compound, provide strong support for the above. In the studies reported here, a model of the enzyme in complex with triclosan and the cofactor NAD has been built by homology modeling with a view to understand its binding properties and to explore the potential of triclosan as a lead compound in designing effective antimalarial drugs. The model indeed provided the structural rationale for its interaction with ligands and the cofactor and revealed unique characteristics of its binding site which could be exploited for improving the specificity of the inhibitors.     

Multiple triclosan targets in Trypanosoma brucei

Trypanosoma brucei genes encoding putative fatty acid synthesis enzymes are homologous to those encoding type II enzymes found in bacteria and organelles such as chloroplasts and mitochondria. It was therefore not surprising that triclosan, an inhibitor of type II enoyl-acyl carrier protein (enoyl-ACP) reductase, killed both procyclic forms and bloodstream forms of T. brucei in culture with 50% effective concentrations (EC(50)s) of 10 and 13 microM, respectively. Triclosan also inhibited cell-free fatty acid synthesis, though much higher concentrations were required (EC(50)s of 100 to 200 microM). Unexpectedly, 100 microM triclosan did not affect the elongation of [(3)H]laurate (C(12:0)) to myristate (C(14:0)) in cultured bloodstream form parasites, suggesting that triclosan killing of trypanosomes may not be through specific inhibition of enoyl-ACP reductase but through some other mechanism. Interestingly, 100 microM triclosan did reduce the level of incorporation of [(3)H]myristate into glycosyl phosphatidylinositol species (GPIs). Furthermore, we found that triclosan inhibited fatty acid remodeling in a cell-free assay in the same concentration range required for killing T. brucei in culture. In addition, we found that a similar concentration of triclosan also inhibited the myristate exchange pathway, which resides in a distinct subcellular compartment. However, GPI myristoylation and myristate exchange are specific to the bloodstream form parasite, yet triclosan kills both the bloodstream and procyclic forms. Therefore, triclosan killing may be due to a nonspecific perturbation of subcellular membrane structure leading to dysfunction in sensitive membrane-resident biochemical pathways.     

Characterization of the flavin reductase gene (fre) of Escherichia coli and construction of a plasmid for overproduction of the enzyme.

The enzyme NAD(P)H:flavin oxidoreductase (flavin reductase) catalyzes the reduction of soluble flavins by reduced pyridine nucleotides. In Escherichia coli it is part of a multienzyme system that reduces the Fe(III) center of ribonucleotide reductase to Fe(II) and thereby sets the stage for the generation by dioxygen of a free tyrosyl radical required for enzyme activity. Similar enzymes are known in other organisms and may more generally be involved in iron metabolism. We have now isolated the gene for the E. coli flavin reductase from a lambda gt11 library. After DNA sequencing we found an open reading frame coding for a polypeptide of 233 amino acids, with a molecular weight of 26,212 and with an N-terminal segment identical to that determined by direct Edman degradation. The coding sequence is preceded by a weak ribosome binding site centered 8 nucleotides from the start codon and by a promoterlike sequence centered at a distance of 83 nucleotides. In a Kohara library the gene hybridized to position 3680 on the physical map of E. coli. A bacterial strain that overproduced the enzyme approximately 100-fold was constructed. The translated amino acid sequence contained a potential pyridine nucleotide-binding site and showed 25% identity with the C-terminal part of one subunit (protein C) of methane monooxygenase from methanotropic bacteria that reduces the iron center of a second subunit (protein A) of the oxygenase by pyridine nucleotides.     

Effect of substrate binding loop mutations on the structure, kinetics, and inhibition of enoyl acyl carrier protein reductase from Plasmodium falciparum

Enoyl acyl carrier protein reductase (ENR), which catalyzes the final and rate limiting step of fatty acid elongation, has been validated as a potential drug target. Triclosan is known to be an effective inhibitor for this enzyme. We mutated the substrate binding site residue Ala372 of the ENR of Plasmodium falciparum (PfENR) to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli ENR (EcENR) which has a Methionine at the structurally similar position of Ala372 of PfENR. Kinetic studies of the mutants of PfENR and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a threefold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. We generated a second set of mutants to check this hypothesis. These mutants showed loss of function, except in one case, where the crystal structure showed that the substrate binding loop is stabilized by a water bridge network.