An Approach to Identifying Novel Substrates of Bacterial Arylamine N-Acetyltransferases Edward W. Brooke, a Stephen G. Davies, b, * Andrew W. Mulvaney, b Fre´de´rique Pompeo, a Edith Sim a and Richard J. Vickers b a Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK b The Dyson Perrins Laboratory, University of Oxford, South Parks Rd, Oxford OX1 3QY, UK Received 1 October 2002; accepted 3 December 2002 Abstract—Arylamine N-acetyltransferases (NATs) catalyse the acetylation of arylamine, arylhydrazine and arylhydroxylamine substrates by acetyl Coenzyme A. NAT has been discovered in a wide range of eukaryotic and prokaryotic species. Although pro- karyotic NATs have been implicated in xenobiotic metabolism, to date no endogenous role has been identified for the arylamine N-acetyl transfer reaction in prokaryotes. Investigating the substrate specificity of these enzymes is one approach to determining a possible endogenous role for prokaryotic NATs. We describe an accurate and efficient assay for NAT activity that is suitable for high-throughput screening of potential NAT ligands. This assay has been utilised to identify novel substrates for pure NAT from Salmonella typhimurium and Mycobacterium smegmatis which show a relationship between the lipophilicity of the arylamine and its activity as a substrate. The lipophilic structure/activity relationship observed is proposed to depend on the topology of the active site using docking studies of the crystal structures of these NAT isoenzymes. The evidence suggests an endogenous role of NAT in the protection of bacteria from aromatic and lipophilic toxins. # 2002 Elsevier Science Ltd. All rights reserved. Introduction Arylamine N-acetyltransferases (NATs) are poly- morphic xenobiotic metabolizing enzymes first identi- fied in humans as responsible for the inactivation of the front-line anti-tubercular drug isoniazid (INH). 1 NATs catalyze the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to arylamine, hydrazine 2 and arylhydroxylamine substrates 3 (Scheme 1) including activated carcinogens. Interindividual variability in rates of drug metabolism 2,4,5 and susceptibility to ary- lamine carcinogens 6,7 have been linked to polymorph- isms in the two active human isoenzymes NAT1 and NAT2. 8 NAT has also been identified in the bacterium Salmo- nella typhimurium (STNAT), as a factor influencing carcinogen susceptibility in mutagenicity testing. 9 As bacterial genomes have become available, NAT has been discovered in more than 20 prokaryotes including Mycobacterium tuberculosis. 10 A NAT homologue, rifa- mycin amide synthetase, 11 has been found in Amycola- topsis mediterranei where it is involved in biosynthesis of the natural antibiotic rifamycin. 12 The nat gene in M. tuberculosis appears in a putative operon that encodes for enzymes capable of metabolizing aromatic com- pounds and suggests a role for mycobacterial NAT in xenobiotic metabolism. 10 The mycobacterial NATs acetylate and hence inactivate isoniazid which could have important implications for isoniazid resistance in M. tuberculosis. 13 Overexpression of NAT from M. tuberculosis in M. smegmatis has resulted in an increased resistance of the recombinant mycobacterium to isoniazid while NAT knockout strains have been shown to exhibit increased sensitivity to INH. 14 0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0968-0896(02)00642-9 Bioorganic & Medicinal Chemistry 11 (2003) 1227–1234 Scheme 1. *Corresponding author. Tel.: +44-1865-275680; fax: +44-1865- 275674; e-mail: steve.davies@chem.ox.ac.uk