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一种用于筛选和鉴定新型蛋白质N-乙酰转移酶的连续检测方法揭示了水稻通用控制非抑制性5相关N-乙酰转移酶2的活性。

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

作者信息

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

机构信息

Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France.

出版信息

Front Plant Sci. 2022 Feb 22;13:832144. doi: 10.3389/fpls.2022.832144. eCollection 2022.

DOI:10.3389/fpls.2022.832144
PMID:35273627
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8902505/
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 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 (), OsGNAT2. We took advantage of an medium-scale characterization of OsGNAT2 specificity to identify and then validate 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.

摘要

蛋白质N - 乙酰基转移酶（NATs）属于一般控制非抑制性5（Gcn5）相关的N - 乙酰基转移酶（GNATs）超家族。GNATs催化乙酰基从乙酰辅酶A转移到多种受体的反应性胺部分。NAT序列很难与GNAT超家族的其他成员区分开来，并且有许多未表征的GNATs。为了促进新GNATs的发现和表征，我们开发了一种新的连续非放射性测定法。该测定法几乎与底物无关，可用于获得底物特异性线索。我们首先用表征良好的NatA（SpNatA）验证了该测定法。用各种肽测定了SpNatA的动力学参数，证实了新测定法的稳健性。我们发现肽底物越长，酶的效率越高。作为新测定法相关性的概念验证，我们表征了水稻的一个NAA90成员，即OsGNAT2。我们利用对OsGNAT2特异性的中等规模表征来鉴定并随后验证了几种特异性肽底物。通过该测定法，我们揭示了碱性残基对OsGNAT2活性的远程协同作用。总体而言，这种新的高通量测定法有助于更好地理解任何GNAT的底物特异性和活性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee58/8902505/c01bf087ce3e/fpls-13-832144-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee58/8902505/f512f8b2c7c0/fpls-13-832144-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee58/8902505/346cf5688db1/fpls-13-832144-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee58/8902505/d536f3a6a76c/fpls-13-832144-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee58/8902505/8c5456d44826/fpls-13-832144-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee58/8902505/c01bf087ce3e/fpls-13-832144-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee58/8902505/f512f8b2c7c0/fpls-13-832144-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee58/8902505/346cf5688db1/fpls-13-832144-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee58/8902505/d536f3a6a76c/fpls-13-832144-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee58/8902505/8c5456d44826/fpls-13-832144-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee58/8902505/c01bf087ce3e/fpls-13-832144-g005.jpg

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