Human Acetyl CoA:Arylamine<i>N-</i>Acetyltransferase Variants Generated by Random Mutagenesis

Summerscales, Joanna; Josephy, P. David

doi:10.1124/mol.65.1.220

Cited by 16 publications

(10 citation statements)

References 32 publications

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“…Human skin also bears high activity of cytosolic N ‐acetylation for detoxification of, for example, arylamines or hydrazines (45,46) and, therefore, provides ‘first‐pass’ metabolism in skin (47). Unlike the NAT2 enzyme, which is found predominantly in the liver and GI tract, NAT1 is distributed widely in extrahepatic tissues of the body including skin (28,48).…”

Section: Discussionmentioning

confidence: 99%

Xenobiotic metabolism capacities of human skin in comparison with a 3D‐epidermis model and keratinocyte‐based cell culture as in vitro alternatives for chemical testing: phase II enzymes

Götz

Pfeiffer

Tigges

et al. 2012

Experimental Dermatology

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The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first‐pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic‐metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S‐transferase, UDP‐glucuronosyltransferase and N‐acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI‐200), immortalized keratinocyte‐based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI‐200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing.

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Section: Discussionmentioning

confidence: 99%

Xenobiotic metabolism capacities of human skin in comparison with a 3D‐epidermis model and keratinocyte‐based cell culture as in vitro alternatives for chemical testing: phase II enzymes

Götz

Pfeiffer

Tigges

et al. 2012

Experimental Dermatology

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“…Various mutagenesis studies and kinetic analyses have been conducted to determine specific regions or residues involved in substrate binding and selectivity, and structural stability. Such studies include the functional analysis of recombinant human NAT1/NAT2 chimeras [49], sitedirected mutagenesis of residues 125, 127 and 129 in human NAT1 and NAT2 [50], as well as random mutagenesis of human NAT2 [51].…”

Section: Eukaryotic Natsmentioning

confidence: 99%

Structure and Mechanism of Arylamine N-Acetyltransferases

Westwood¹,

Kawamura²,

Fullam³

et al. 2006

CTMC

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Arylamine N-acetyltransferases (NATs) are a family of phase II drug-metabolising enzymes which are important in the biotransformation of various aromatic and heterocyclic amines and hydroxylamines, arylhydrazines and arylhydrazides. NATs are present in a wide range of eukaryotes and prokaryotes. Humans have two functional NAT isoforms, both of which are highly polymorphic. The pharmacogenetics of NATs is an area which has been extensively studied. The determination of the X-ray crystal structure of NAT from Salmonella typhimurium led to the identification of the catalytically essential triad of residues: Cys-His-Asp, which is present in all functional NAT enzymes. Recent co-crystallisation data and in silico docking studies of NAT from Mycobacterium smegmatis with substrates and inhibitors have aided the identification of important contact residues within the active site. The X-ray crystal structures of four prokaryotic NAT proteins have now been determined, and these have been used to generate structural models of eukaryotic NATs, providing valuable insight into their active-site architecture. In addition to aiding crystallographic experiments, recent progress in the production of recombinant prokaryotic and eukaryotic NATs has allowed comparative studies of the kinetics and activity profiles of these enzymes. In this review we present an overview of recent structural and activity studies on NAT enzymes, and we outline how in silico methods may be used to predict NAT protein-ligand interactions based on the current knowledge.

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“…It has also been demonstrated using random [40] and sitedirected [41] mutagenesis that a range of amino acid substitutions can result in a decrease in activity despite a lack of effect on protein stability. These point mutations, resulting in substitution of a particular amino acid, are likely to be specific effects, rather than due to non-specific structural modifications resulting in poorly folded, and hence unstable, proteins.…”

Section: Modification Of the Putative Acetyl-coa Binding Sitementioning

confidence: 99%

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

Sandy

Mushtaq

Holton

et al. 2005

Biochemical Journal

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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.

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Human Acetyl CoA:ArylamineN-Acetyltransferase Variants Generated by Random Mutagenesis

Cited by 16 publications

References 32 publications

Xenobiotic metabolism capacities of human skin in comparison with a 3D‐epidermis model and keratinocyte‐based cell culture as in vitro alternatives for chemical testing: phase II enzymes

Xenobiotic metabolism capacities of human skin in comparison with a 3D‐epidermis model and keratinocyte‐based cell culture as in vitro alternatives for chemical testing: phase II enzymes

Structure and Mechanism of Arylamine N-Acetyltransferases

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

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