The Structure of Arylamine N-acetyltransferase from Mycobacterium smegmatis—An Enzyme which Inactivates the Anti-tubercular Drug, Isoniazid

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Abstract

Arylamine N-acetyltransferases which acetylate and inactivate isoniazid, an anti-tubercular drug, are found in mycobacteria including Mycobacterium smegmatis and Mycobacterium tuberculosis. We have solved the structure of arylamine N-acetyltransferase from M. smegmatis at a resolution of 1.7 Å as a model for the highly homologous NAT from M. tuberculosis. The fold closely resembles that of NAT from Salmonella typhimurium, with a common catalytic triad and domain structure that is similar to certain cysteine proteases. The detailed geometry of the catalytic triad is typical of enzymes which use primary alcohols or thiols as activated nucleophiles. Thermal mobility and structural variations identify parts of NAT which might undergo conformational changes during catalysis. Sequence conservation among eubacterial NATs is restricted to structural residues of the protein core, as well as the active site and a hinge that connects the first two domains of the NAT structure. The structure of M. smegmatis NAT provides a template for modelling the structure of the M. tuberculosis enzyme and for structure-based ligand design as an approach to designing anti-TB drugs.

Introduction

Arylamine N-acetyltransferase activity was first identified as the enzymic activity in humans responsible for the inactivation of isoniazid, the major anti-tubercular agent.1 Arylamine N-acetyltransferases are now known to constitute a major family of enzymes which acetylate a range of arylamine, arylhydroxylamines and arylhydrazines using acetyl-CoA as the acetyl donor.2., 3. Other members of the family, including the terminal protein in the cluster of enzymes responsible for rifamycin synthesis,4 are likely to be involved in amide bond formation using a different acyl donor. It is therefore possible that individual members of the NAT family provide a range of activities in the different organisms in which they are found. Mycobacteria are distinguished by distinctive metabolism resulting in the synthesis of mycolic acids, long chain fatty acids that form a layer of the tough mycobacterial cell wall. A further distinctive feature of mycobacteria is their unique sensitivity to isoniazid.5 Isoniazid inhibits mycolic acid synthesis. However, isoniazid is a pro-drug and must be activated by oxidation. The gene product of katG, which has both catalase and peroxidase activity associated with it, activates isoniazid.6., 7. The activated drug is thought to exert its effect, at least partially, through inhibition of an enoyl-acyl carrier protein (ACP) reductase (InhA).8 This is an enzyme of the multicomponent fatty acid synthase type II (FAS II) complex, involved in mycolic acid synthesis. Another component of FAS II, a β-ketoacyl ACP synthase (KasA) has also been identified as a target.9 If isoniazid is acetylated by NAT, the product acetylisoniazid cannot be oxidized to its active form by katG. The gene for NAT is present in the mycobacterial species Mycobacterium tuberculosis,10 M. bovis bacilli Calmette-Guérin (BCG), Mycobacterium avium and Mycobacterium smegmatis.11 When the gene for M. tuberculosis NAT is over-expressed in M. smegmatis the result is that resistance to isoniazid is increased,12 and, as would be expected, knocking out the nat gene increases the sensitivity of M. smegmatis to isoniazid.13 It is unclear whether NAT from mycobacteria also has an endogenous role, although knocking out the nat gene from M. smegmatis increases the duration of the lag phase of bacterial cell growth.13 Therefore, on the basis of its participation in isoniazid metabolism, and the effect of knocking out the nat gene in M. smegmatis, NAT from M. tuberculosis is a strong candidate as a target for anti-tubercular therapy. In order to apply an approach of rational drug design, the structure of NAT from a mycobacterium is required. The crystal structure of NAT from Salmonella typhimurium has been obtained at 2.8 Å resolution.14 This was the first member of the NAT family for which a crystal structure has been determined. NAT from S. typhimurium is only 32% identical to NAT from M. tuberculosis at the amino acid level. Therefore, it has been important to determine the structure of a mycobacterial NAT. The NAT from M. smegmatis is 60% identical to the NAT from M. tuberculosis, and both enzymes are able to acetylate isoniazid.15 M. smegmatis NAT, in contrast to M. tuberculosis NAT, when expressed as a recombinant protein in Escherichia coli, is highly soluble, and crystallises readily. We have therefore been able to determine the crystal structure of M. smegmatis NAT at a resolution of 1.7 Å.

Section snippets

Purification and characterisation

The NAT from M. smegmatis has been generated as a recombinant soluble protein with an N-terminal hexahistidine tag in E. coli. The protein has been purified to homogeneity on a nickel affinity resin and the tag has been removed with thrombin. Thrombin cleavage leaves three additional non-authentic residues (a glutamic acid, a serine and a histidine) at the amino terminus. The pure protein is active and catalyses the acetylation of isoniazid (Km, 87((±18) μM; Vmax, 115(±33) nmol min−1 mg−1 protein) as

Dimerisation

M. smegmatis NAT forms dimers in all of the crystal forms analysed here through an edge-to-edge β-sheet association involving the third domain. This region forms a similar dimer interface in crystals of S. typhimurium NAT, and is well conserved among eukaryotic (but not prokaryotic) NAT enzymes. It has been observed among certain NAT isozymes that NAT forms dimers under conditions of catalysis,20 and it may be that the third domain is responsible for such oligomerisation.

Catalytic triad

The geometry of the

Experimental

All chemicals were purchased from Sigma-Aldrich unless indicated otherwise.

Acknowledgements

We are grateful to the Wellcome Trust for continued financial support. A.M. is in receipt of an MRC studentship. We thank Anna Upton and Mark Payton for helpful discussions. We would also thank the staff of the ESRF and SRS synchrotrons.

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