title: Arylamine N-acetyltransferase of Mycobacterium tuberculosis is a polymorphic enzyme and a site of isoniazid metabolism.
abstract: Arylamine N-acetyltransferases (NATs; E.C 2.3.1.5) N-acetylate arylhydralazine and arylamine substrates using acetyl coenzyme A. Human NAT2 acetylates and inactivates the antituberculosis drug, isoniazid (INH), and is polymorphic. We previously demonstrated that there is a homologue of human NAT2 in Mycobacterium tuberculosis, whose product N-acetylates INH in vitro. We now demonstrate that the nat gene is expressed in M. tuberculosis and M. bovis Bacille Calmette-Guerin (BCG), using reverse transcription-polymerase chain reaction and Western blotting. The NAT protein is active in M. bovis BCG in vivo, as detected by the presence of N-acetyl INH in M. bovis BCG lysates grown in INH. Sequence analysis of the M. tuberculosis nat coding region reveals a single nucleotide polymorphism in 18% of a random cohort of M. tuberculosis clinical isolates, conferring a G to R change. The recombinant mutant protein appears less stable than the wild type, and has an apparent affinity for INH of 10-fold less than the wild type. Modelling the change in M. tuberculosis NAT shows that the G to R change is close to the active site, and supports the experimental findings. Minimum inhibitory concentration data suggest that this polymorphism in nat is linked to low-level changes in the INH susceptibility of M. tuberculosis clinical isolates.
authors.affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK. anna.upton@pharm.ox.ac.uk
authors.forename: A M
authors.initials: AM
authors.lastname: Upton
authors.forename: A
authors.initials: A
authors.lastname: Mushtaq
authors.forename: T C
authors.initials: TC
authors.lastname: Victor
authors.forename: S L
authors.initials: SL
authors.lastname: Sampson
authors.forename: J
authors.initials: J
authors.lastname: Sandy
authors.forename: D M
authors.initials: DM
authors.lastname: Smith
authors.forename: P V
authors.initials: PV
authors.lastname: van Helden
authors.forename: E
authors.initials: E
authors.lastname: Sim
mesh_terms: D000107:Acetylation; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015153:Blotting, Western; D006801:Humans; D007538:Isoniazid / Q000378:metabolism* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D009163:Mycobacterium bovis; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D011110:Polymorphism, Genetic / Q000235:genetics*; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; D001191:Arylamine N-Acetyltransferase; C478900:NAT2 protein, human; D007538:Isoniazid