The COOH Terminus of Arylamine N-Acetyltransferase from Salmonella typhimurium Controls Enzymic Activity

Adeel Mushtaq,Mark Payton,Edith Sim

doi:10.1074/jbc.m104365200

Abstract

Arylamine N-acetyltransferases (NATs) are a homologous family of enzymes, which acetylate arylamines, arylhydroxylamines, and arylhydrazines by acetyl transfer from acetyl-coenzyme A (Ac-CoA) and are found in many organisms. NAT was first identified as the enzyme responsible for the inactivation of the anti-tubercular drug isoniazid in humans. The three-dimensional structure of NAT from Salmonella typhimurium has been resolved and shown to have three distinct domains and an active site catalytic triad composed of "Cys(69)-His(107)-Asp(122)," which is typical of hydrolytic enzymes such as the cysteine proteases. The crystal unit cell consists of a dimer of tetramers, with the C terminus of individual monomers juxtaposed. To investigate the function of the first two domains of full-length NAT from S. typhimurium and to investigate the role of the C terminus of NAT, truncation mutants were made with either the C-terminal undecapeptide or the entire third domain (85 amino acids) missing. Unlike the full-length NAT protein (281 amino acids), the truncation mutants of NAT from S. typhimurium are toxic when overexpressed intracellularly in Escherichia coli. Full-length NAT hydrolyses Ac-CoA but only in the presence of an arylamine substrate. Both truncation mutants, however, hydrolyze Ac-CoA even in the absence of arylamine substrate, illustrating that the C-terminal undecapeptide controls hydrolysis of Ac-CoA by NAT from S. typhimurium.

Highlights

Arylamine N-acetyltransferases (NATs,1 EC 2.3.1.5) vary in size from 30 to 34 kDa and form a distinct protein family (1)
NAT from Salmonella typhimurium was first identified from strains of the bacterium used in the Ames test for carcinogens (14) and it was subsequently shown to be involved in the activation of hydroxyarylamine carcinogens by O-acetylation (2, 15, 16)
We have investigated the function of the first two domains of full-length NAT from S. typhimurium, which corresponds to 196 amino acid residues, and the role of the C terminus of NAT, by making truncation mutants with either the C-terminal undecapeptide (AAFDTHPEAGK) or the entire third domain missing (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Cloning—Fragments of the gene encoding full-length S. typhimurium NAT were cloned following amplification of the regions from the open reading frame of NAT in genomic DNA from S. typhimurium (Strain L2, from Jay Hinton, IMM, Oxford). The nst-196,nst-270 fragments and the open reading frame of S. typhimurium NAT, inclusive of a N-terminal hexa-histidine tag (nst-281) were further subcloned from pET28b(ϩ), into the vector pBAD/gIII (24) (Invitrogen Inc., Groningen, Holland) using the NcoI and XhoI sites, followed by transformation of the pBAD/ gIII vectors into E. coli strain TOP 10 (Invitrogen), for propagation and expression. Protein concentrations of soluble fractions were determined by measuring the absorbance at 280 nm or by using the Bradford assay (28). NAT protein in insoluble fractions was quantitated following Western blotting by comparison with known amounts of soluble pure NST-281. The enzyme solution (final volume 1 ml) contained between 50 pg and 1 ␮g of pure protein in 20 mM Tris-HCl and 1 mM EDTA at pH 7.5 with the addition of 2 mM INH as required. The lowest energy docking solutions were viewed and analyzed using SPDBV (39)

RESULTS

ND ND

DISCUSSION