Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines

Abstract

The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-HisAsp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly 126 , implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 Å (where 1 Å = 0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity.

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

“…There is an extensive history of using information from bacterial enzymes to understand the interactions of human enzymes with xenobiotics, e.g., cytochrome P450s (Poulos et al, 1985) and more recently the arylamine N-acetyl transferase, enzymes (Sinclair et al, 2000). The use of a series of bacterial enzymes has proved particularly instructive (Lewis, 2001;Sandy et al, 2005).…”

Purification, crystallization and preliminary X-ray characterization ofBacillus cereusarylamineN-acetyltransferase 3 [(BACCR)NAT3]

“…Surprisingly, mutation of Asp122 to Glu122 resulted in an enzyme with very low activity (more than 40-fold less active than the reference enzyme; Zang et al, 2007). Although the Asp residue is highly conserved and is present in the majority of NAT sequences, certain NAT orthologues from Bacillus species have been shown to possess a Glu residue instead of the canonical Asp in their catalytic triad (Sandy et al, 2005;Pluvinage et al, 2007). In B. cereus, one of the three NAT isoenzymes, (BACCR)NAT3, has a Glu residue in its catalytic triad (Glenn et al, 2011) but nonetheless appears to be as active as other known NAT enzymes towards several prototypic NAT substrates (unpublished results).…”

“…The crystal structures of several NAT enzymes and mutational studies have underlined the importance of the cysteine-protease-like catalytic triad (Cys-His-Asp; Sim, Walters et al, 2008;Sinclair et al, 2000). Substitution of the Cys or His residues of the triad leads to a complete loss of enzyme activity (Sandy et al, 2005). Recently, studies of a human NAT2 single-nucleotide polymorphism (NAT2*12D) have shown that substitution of the catalytic triad residue Asp122 can have a dramatic impact on the activity of the enzyme (Zang et al, 2007).…”

<em>In Silico</em> Screening and Analysis of Potential Inhibitors of Arylamine N-Acetyltransferases (NATs) from the Traditional Chinese Medicine: A Study Using Free Available Tools

“…In fact, the fold of human NAT1 and NAT2 closely resembles the overall structure of TBNAT [11,20,21]. NATs overall fold is composed of three independent domains of approximately the same length.…”