Mapping roles of active site residues in the acceptor site of the PA3944 Gcn5-related N-acetyltransferase enzyme.

Autor: Variot C; Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California, USA., Capule D; Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California, USA., Arolli X; Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, Illinois, USA., Baumgartner J; Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California, USA., Reidl C; Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, Illinois, USA., Houseman C; Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California, USA., Ballicora MA; Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, Illinois, USA., Becker DP; Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, Illinois, USA., Kuhn ML; Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California, USA.
Jazyk: angličtina
Zdroj: Protein science : a publication of the Protein Society [Protein Sci] 2023 Aug; Vol. 32 (8), pp. e4725.
DOI: 10.1002/pro.4725
Abstrakt: An increased understanding of how the acceptor site in Gcn5-related N-acetyltransferase (GNAT) enzymes recognizes various substrates provides important clues for GNAT functional annotation and their use as chemical tools. In this study, we explored how the PA3944 enzyme from Pseudomonas aeruginosa recognizes three different acceptor substrates, including aspartame, NANMO, and polymyxin B, and identified acceptor residues that are critical for substrate specificity. To achieve this, we performed a series of molecular docking simulations and tested methods to identify acceptor substrate binding modes that are catalytically relevant. We found that traditional selection of best docking poses by lowest S scores did not reveal acceptor substrate binding modes that were generally close enough to the donor for productive acetylation. Instead, sorting poses based on distance between the acceptor amine nitrogen atom and donor carbonyl carbon atom placed these acceptor substrates near residues that contribute to substrate specificity and catalysis. To assess whether these residues are indeed contributors to substrate specificity, we mutated seven amino acid residues to alanine and determined their kinetic parameters. We identified several residues that improved the apparent affinity and catalytic efficiency of PA3944, especially for NANMO and/or polymyxin B. Additionally, one mutant (R106A) exhibited substrate inhibition toward NANMO, and we propose scenarios for the cause of this inhibition based on additional substrate docking studies with R106A. Ultimately, we propose that this residue is a key gatekeeper between the acceptor and donor sites by restricting and orienting the acceptor substrate within the acceptor site.
(© 2023 The Protein Society.)
Databáze: MEDLINE
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