Arylamine N-acetyltransferase of Mycobacterium Tuberculosis Is a Polymorphic Enzyme and a Site of Isoniazid Metabolism

Mol Microbiol. 2001 Oct;42(2):309-17. doi: 10.1046/j.1365-2958.2001.02648.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Arylamine N-Acetyltransferase / chemistry
  • Arylamine N-Acetyltransferase / genetics*
  • Arylamine N-Acetyltransferase / metabolism*
  • Blotting, Western
  • Humans
  • Isoniazid / metabolism*
  • Isoniazid / pharmacology
  • Microbial Sensitivity Tests
  • Models, Molecular
  • Mycobacterium bovis
  • Mycobacterium tuberculosis / drug effects
  • Mycobacterium tuberculosis / enzymology*
  • Mycobacterium tuberculosis / genetics
  • Phylogeny
  • Polymorphism, Genetic / genetics*
  • Protein Conformation
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • RNA, Messenger
  • Recombinant Proteins
  • Arylamine N-Acetyltransferase
  • NAT2 protein, human
  • Isoniazid