Journal of Molecular Biology
The Structure of Arylamine N-acetyltransferase from Mycobacterium smegmatis—An Enzyme which Inactivates the Anti-tubercular Drug, Isoniazid
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.
References (49)
- et al.
Lessons from the rifamycin biosynthetic gene cluster
Curr. Opin. Chem. Biol.
(1999) - et al.
The genetics and biochemistry of isoniazid resistance in Mycobacterium tuberculosis
Microbes Infect.
(2000) - et al.
Purification, characterization, and crystallization of an N-hydroxyarylamine O-acetyltransferase from Salmonella typhimurium
Protein Expt. Purif.
(1998) - et al.
Arylamine N-acetyltransferase from fast (C57BL6) and slow (A/J) N-acetylating strains of mice
Biochem. Pharmacol.
(1990) - et al.
Drug metabolising N-acetyltransferase activity in human cell lines
Biochim. Biophys. Acta
(1991) - et al.
Placental arylamine N-acetyltransferase type 1: potential contributory source of urinary folate catabolite p-acetamidobenzoylglutamate during pregnancy
Biochim. Biophys. Acta
(2000) - et al.
Purification and physical–chemical properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver
Biochim. Biophys. Acta
(1983) - et al.
Catalytic triads and their relatives
Trends Biochem. Sci.
(1998) - et al.
Protein structure comparison by alignment of distance matrices
J. Mol. Biol.
(1993) Structure of the epididymal retinoic acid binding protein at 2.1 Å resolution
Structure
(1993)
The crystal structure of cruzain: a therapeutic target for Chagas’ disease
J. Mol. Biol.
Crystal structure of human bleomycin hydrolase, a self-compartmentalizing cysteine protease
Struct. Fold Des.
Identification of the calcium binding site and a novel ytterbium site in blood coagulation factor XIII by X-ray crystallography
J. Biol. Chem.
A fragment consisting of the first 204 amino-terminal amino acids of human arylamine N-acetyltransferase one (NAT1) and the first transacetylation step of catalysis
Biochem. Pharmacol.
Acetyl-coenzyme A: arylamine N-acetyltransferase. Role of the acetyl-enzyme intermediate and the effects of substituents on the rate
J. Biol. Chem.
Molecular mechanisms of isoniazid: a drug at the front line of tuberculosis control
Trends Microbiol.
High-performance liquid chromatographic analysis of isoniazid and acetylisoniazid in biological fluids
J. Chromatog.
Definition of general topological equivalence in protein structures. A procedure involving comparison of properties and relationships through simulated annealing and dynamic programming
J. Mol. Biol.
Comparative protein modelling by satisfaction of spatial restraints
J. Mol. Biol.
Genetic control of isoniazid metabolism in man
Brit. Med. J.
N-Acetylation pharmacogenetics
Pharmacol. Rev.
An update on genetic, structural and functional studies of arylamine N-acetyltransferases in eucaryotes and procaryotes
Hum. Mol. Genet.
Strategies for new drug development
The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis
Nature
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