自由基S-腺苷甲硫氨酸酶的机制研究：丙酮酸甲酸裂解酶激活酶和赖氨酸2,3-氨基变位酶案例研究

Mechanistic Studies of Radical SAM Enzymes: Pyruvate Formate-Lyase Activating Enzyme and Lysine 2,3-Aminomutase Case Studies.

作者信息

Byer Amanda S, McDaniel Elizabeth C, Impano Stella, Broderick William E, Broderick Joan B

机构信息

Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, United States.

出版信息

Methods Enzymol. 2018;606:269-318. doi: 10.1016/bs.mie.2018.04.013. Epub 2018 Jul 7.

The radical SAM enzyme superfamily is large and diverse, with ever-increasing numbers of examples of characterized reactions. This chapter focuses on the methodology we have developed over the last 25 years for working with these enzymes, with the specific examples discussed being the pyruvate formate-lyase activating enzyme (PFL-AE) and lysine 2,3-aminomutase (LAM). Both enzymes are purified from overexpressing Escherichia coli, but differ in that PFL-AE is expressed without an affinity tag and does not require iron-sulfur cluster reconstitution, while LAM purification is carried out through use of a His affinity tag and the enzyme benefits from cluster reconstitution. Because of radical SAM enzymes' catalytic need for a [4Fe-4S] cluster, we present methods for characterization and incorporation of a full [4Fe-4S] cluster in addition to enzyme activity assay protocols. Synthesis of SAM (S-adenosyl-l-methionine) and its analogs have played an important role in our mechanistic studies of radical SAM enzymes, and their synthetic methods are also presented in detail.

自由基S-腺苷甲硫氨酸（SAM）酶超家族庞大且多样，其已被表征反应的实例数量不断增加。本章重点介绍我们在过去25年中开发的用于研究这些酶的方法，所讨论的具体实例为丙酮酸甲酸裂解酶激活酶（PFL-AE）和赖氨酸2,3-氨基变位酶（LAM）。这两种酶均从过表达的大肠杆菌中纯化得到，但不同之处在于，PFL-AE表达时没有亲和标签，且不需要铁硫簇重构，而LAM的纯化是通过使用His亲和标签进行的，并且该酶受益于簇重构。由于自由基SAM酶催化需要[4Fe-4S]簇，我们除了介绍酶活性测定方案外，还介绍了[4Fe-4S]簇的表征和掺入方法。SAM（S-腺苷-L-甲硫氨酸）及其类似物的合成在我们对自由基SAM酶的机理研究中发挥了重要作用，其合成方法也将详细介绍。