精氨酰转移酶（ATE1）铁硫簇的重构

Reconstitution of the Arginyltransferase (ATE1) Iron-Sulfur Cluster.

作者信息

Van Verna, Smith Aaron T

机构信息

Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD, USA.

出版信息

Methods Mol Biol. 2023;2620:209-217. doi: 10.1007/978-1-0716-2942-0_23.

DOI:10.1007/978-1-0716-2942-0_23

PMID:37010764

Abstract

As global regulators of eukaryotic homeostasis, arginyltransferases (ATE1s) have essential functions within the cell. Thus, the regulation of ATE1 is paramount. It was previously postulated that ATE1 was a hemoprotein and that heme was an operative cofactor responsible for enzymatic regulation and inactivation. However, we have recently shown that ATE1 instead binds an iron-sulfur ([Fe-S]) cluster that appears to function as an oxygen sensor to regulate ATE1 activity. As this cofactor is oxygen-sensitive, purification of ATE1 in the presence of O results in cluster decomposition and loss. Here, we describe an anoxic chemical reconstitution protocol to assemble the [Fe-S] cluster cofactor in Saccharomyces cerevisiae ATE1 (ScATE1) and Mus musculus ATE1 isoform 1 (MmATE1-1).

摘要

作为真核生物体内稳态的全局调节因子，精氨酰转移酶（ATE1s）在细胞内具有重要功能。因此，对ATE1的调节至关重要。此前曾推测ATE1是一种血红素蛋白，血红素是负责酶调节和失活的有效辅因子。然而，我们最近发现ATE1反而结合了一个铁硫（[Fe-S]）簇，该簇似乎作为氧传感器来调节ATE1活性。由于这种辅因子对氧敏感，在有氧条件下纯化ATE1会导致簇分解和丢失。在这里，我们描述了一种缺氧化学重组方案，用于在酿酒酵母ATE1（ScATE1）和小家鼠ATE1同工型1（MmATE1-1）中组装[Fe-S]簇辅因子。