Structural investigation of mutant Mycobacterium smegmatis arylamine N-acetyltransferase: a model for a naturally occurring functional polymorphism in Mycobacterium tuberculosis arylamine N-acetyltransferase Akane Kawamura, a James Sandy, a Anna Upton, a Martin Noble, b and Edith Sim a, * a Department of Pharmacology, University of Oxford, Manfield Road, Oxford OX1 3QT, UK b Laboratory of Molecular Biophysics, Department of Biochemistry and Oxford Centre for Molecular Sciences, Oxford University, South Parks Road, Oxford, OX1 3QU, UK Received 2 May 2002, and in revised form 23 August 2002 Abstract Arylamine N-acetyltransferase (NAT) acetylates the front-line anti-tuberculosis drug isoniazid (INH) and has been identified in Mycobacterium tuberculosis. A naturally occurring single nucleotide polymorphism (SNP) was recently found in the NAT gene in clinical isolates of M. tuberculosis. The nucleotide change from G ! A (619) produces an amino acid change Gly 207 Arg, which appears to reduce the activity of the NAT from M. tuberculosis (TBNAT). It has not been possible to generate sufficient soluble recombinant TBNAT for 3D structural studies. Therefore, Mycobacterium smegmatis NAT (SMNAT), which has 60% identity to TBNAT and has Gly at 207, was used as a model to investigate the possible structural effects of the G ! A 619 SNP. The mutant form of SMnat (SM207Rnat) was constructed by in vitro site-directed mutagenesis and was heterologously expressed with an N- terminal His tag in Escherichia coli, for comparison with the SMNAT. Both recombinant SMNATs were purified using Ni affinity chromatography and treated with thrombin to cleave the tag. Both proteins were produced with average yields of over 10 mg/L and were active. Substrate specificity and thermal stability of SM207RNAT were assessed and compared with the wild type SMNAT using kinetic assays and circular dichroism spectroscopy. SM207RNAT was crystallised and a data set of 2.00  A resolution was obtained. The SM207RNAT had different substrate specificities to the wild type protein and the 3D structures revealed that the Gly 207 Arg mutation caused slight changes in the orientation of His 203 in SMNAT. Ó 2002 Elsevier Science (USA). All rights reserved. Arylamine N-acetyltransferases (NATs: EC 2.3.1.5) are cytosolic, drug metabolising enzymes responsible for catalysing the acetylation of arylamines, arylhydroxyl- amines, and arylhydrazine xenobiotics and carcinogens [1] using acetyl coenzyme A as a cofactor [2]. NAT was first identified in humans as a key enzyme in the poly- morphic metabolism of the leading anti-tubercular drug isoniazid (INH) [3]. One of the two human NAT isoen- zymes NAT2 is responsible for inter-individual variation in the INH acetylator status of tuberculosis patients. Alleles conferring fast and slow acetylation phenotype differ by single nucleotide polymorphisms (SNPs) within the coding region of human NAT2 [4,5] and genotype– phenotype correlation is well defined. NATs have since been found in a wide range of eukaryotes [6] and prok- aryotes [7,8], most interestingly in Mycobacterium tu- berculosis, the causative agent of tuberculosis. Mycobacteria show exquisite sensitivity to INH [9] and INH still remains a primary choice of antibiotic treatment against tuberculosis [10]. INH inhibits the biosynthesis of mycolic acids, a critical component of the mycobacterial cell wall [11]. However, the precise molecular mechanism by which INH acts within the mycobacterium is not fully understood. The INH pro- drug is converted into its active oxidised state by cata- lase-peroxidase activity, encoded by katG [12]. The bioactive INH moiety is thought to target the 2-trans enoyl acyl carrier protein (ACP) reductase encoded by inhA and to inhibit an essential step in fatty acid Protein Expression and Purification 27 (2003) 75–84 www.elsevier.com/locate/yprep * Corresponding author. Fax: +44-0-1865-271853. E-mail address: esim@molbiol.ox.ac.uk (E. Sim). 1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1046-5928(02)00592-2