A Continuous Assay Set to Screen and Characterize Novel Protein N-Acetyltransferases Unveils Rice General Control Non-repressible 5-Related N-Acetyltransferase2 Activity.

Thomas Asensio,Jean-Baptiste Boyer,Cyril Dian,Thierry Meinnel,Frédéric Rivière,Carmela Giglione

doi:10.3389/fpls.2022.832144

Abstract

Protein N-acetyltransferases (NATs) belong to the general control non-repressible 5 (Gcn5)-related N-acetyltransferases (GNATs) superfamily. GNATs catalyze the transfer of acetyl from acetyl-CoA to the reactive amine moiety of a wide range of acceptors. NAT sequences are difficult to distinguish from other members of the GNAT superfamily and there are many uncharacterized GNATs. To facilitate the discovery and characterization of new GNATs, we have developed a new continuous, non-radioactive assay. This assay is virtually independent of the substrate and can be used to get substrate specificity hints. We validated first the assay with the well-characterized Schizosaccharomyces pombe NatA (SpNatA). The SpNatA kinetic parameters were determined with various peptides confirming the robustness of the new assay. We reveal that the longer the peptide substrate the more efficient the enzyme. As a proof of concept of the relevance of the new assay, we characterized a NAA90 member from rice (Oryza sativa), OsGNAT2. We took advantage of an in vivo medium-scale characterization of OsGNAT2 specificity to identify and then validate in vitro several specific peptide substrates. With this assay, we reveal long-range synergic effects of basic residues on OsGNAT2 activity. Overall, this new, high-throughput assay allows better understanding of the substrate specificity and activity of any GNAT.

Highlights

Protein modifications involve a huge number (>400) of different chemical groups and acceptor places where the modifiers may act1 (Khoury et al, 2011)
NATs and KNATs all belong to the superfamily of general control non-repressible 5 (GCN5)-related N-acetyltransferases (GNATs), whereas only one group among the three types of KATs corresponds to GNATs; the other KATs reported so far belong to the MYST, and p300/CBP families (Berndsen and Denu, 2008; Drazic et al, 2016)
Protein acetyltransferases of the GNAT family, including NATs, KATs, and KNATs, are difficult to distinguish among them and from the other members of the GNAT superfamily that encompass a number of small metabolites as acetyl receptors, including antibiotics, such as chloramphenicol, amino acids, sugars etc. (Vetting et al, 2005)

Summary

Introduction

Protein modifications involve a huge number (>400) of different chemical groups and acceptor places where the modifiers may act (Khoury et al, 2011). Recent data have revealed that a number of N-acetyltransferases display both specificities (KNATs; Aksnes et al, 2019; Bienvenut et al, 2020; Linster et al, 2020; Giglione and Meinnel, 2021). NATs and KNATs all belong to the superfamily of general control non-repressible 5 (GCN5)-related N-acetyltransferases (GNATs), whereas only one group among the three types of KATs corresponds to GNATs; the other KATs reported so far belong to the MYST, and p300/CBP families (Berndsen and Denu, 2008; Drazic et al, 2016). Protein acetyltransferases of the GNAT family, including NATs, KATs, and KNATs, are difficult to distinguish among them and from the other members of the GNAT superfamily that encompass a number of small metabolites as acetyl receptors, including antibiotics, such as chloramphenicol, amino acids, sugars etc. The characterization of rice serotonin acetyltransferase required the screening of as many as 31 open reading frames in Escherichia coli (Kang et al, 2013)

Methods

Results

Conclusion