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Insights into how protein dynamics affects arylamine N-acetyltransferase catalysis.

ABSTRACT: Arylamine N-acetyltransferases (NATs) detoxify arylamines and hydrazine xenobiotics by catalyzing their N-acetylation, which prevents their bioactivation. Here, we reveal how structural dynamics impact NAT protein function. Our data suggest that there are multiple conformations in the catalytic cavity of hamster NAT2 that exchange on the millisecond time scale and enable NATs to accommodate substrates of varying size. The regions spanning N177-L180 and D285-F288, which form unique structures in mammalian NATs, possess inherent motions on the nanosecond time scale. The latter segment becomes more restricted in its motions upon substrate binding according to our NMR XNOE data. This greater rigidity appears to stem from interactions with the substrate. Finally, NAT acetylation has been suggested to protect these enzymes from ubiquitination. Our NMR data on a catalytically active state of hamster NAT2 suggest that structural rearrangements caused by its acetylation might contribute to this protection.

SUBMITTER: Zhang N

PROVIDER: S-EPMC2748875 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Insights into how protein dynamics affects arylamine N-acetyltransferase catalysis.

Zhang Naixia N Walters Kylie J KJ

Biochemical and biophysical research communications 20090520 3

Arylamine N-acetyltransferases (NATs) detoxify arylamines and hydrazine xenobiotics by catalyzing their N-acetylation, which prevents their bioactivation. Here, we reveal how structural dynamics impact NAT protein function. Our data suggest that there are multiple conformations in the catalytic cavity of hamster NAT2 that exchange on the millisecond time scale and enable NATs to accommodate substrates of varying size. The regions spanning N177-L180 and D285-F288, which form unique structures in ...[more]

PMID: 19463782

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Project description:The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous to (HUMAN)NAT1 and their corresponding proteins are functionally similar, but the relationship between (MOUSE)Nat1 and (HUMAN)NAT2 is less clear-cut.To determine whether the (MOUSE)NAT1 and (HUMAN)NAT2 enzymes are functionally equivalent, we expressed and purified (MOUSE)NAT1*1 and analysed its substrate specificity using a panel of arylamines and hydrazines. To understand how specific residues contribute to substrate selectivity, three site-directed mutants of (MOUSE)NAT2*1 were prepared: these were (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. All three exhibited diminished activity towards "(MOUSE)NAT2-specific" arylamines but were more active against hydrazines than (MOUSE)NAT1*1. The inhibitory and colorimetric properties of a selective naphthoquinone inhibitor of (HUMAN)NAT1 and (MOUSE)NAT2 were investigated.Comparing (MOUSE)NAT1*1 with other mammalian NAT enzymes demonstrated that the substrate profiles of (MOUSE)NAT1 and (HUMAN)NAT2 are less similar than previously believed. Three key residues (F125, R127 and Y129) in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 were required for enzyme inhibition and the associated colour change on naphthoquinone binding. In silico modelling of selective ligands into the appropriate NAT active sites further implicated these residues in substrate and inhibitor specificity in mouse and human NAT isoenzymes.Three non-catalytic residues within (HUMAN)NAT1*4 (F125, R127 and Y129) contribute both to substrate recognition and inhibitor binding by participating in distinctive intermolecular interactions and maintaining the steric conformation of the catalytic pocket. These active site residues contribute to the definition of substrate and inhibitor selectivity, an understanding of which is essential for facilitating the design of second generation (HUMAN)NAT1-selective inhibitors for diagnostic, prognostic and therapeutic purposes. In particular, since the expression of (HUMAN)NAT1 is related to the development and progression of oestrogen-receptor-positive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours.

Dataset Information

Insights into how protein dynamics affects arylamine N-acetyltransferase catalysis.

Publications

Insights into how protein dynamics affects arylamine N-acetyltransferase catalysis.

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