Purification and structural elucidation of a cobalamin-dependent radical SAM enzyme.

Autor:	Dill Z; Department of Chemistry, University of Michigan, Ann Arbor, MI, United States; Program in Chemical Biology, University of Michigan, Ann Arbor, MI, United States., Li B; Department of Chemistry, University of Michigan, Ann Arbor, MI, United States., Bridwell-Rabb J; Department of Chemistry, University of Michigan, Ann Arbor, MI, United States; Program in Chemical Biology, University of Michigan, Ann Arbor, MI, United States. Electronic address: jebridwe@umich.edu.
Jazyk:	angličtina
Zdroj:	Methods in enzymology [Methods Enzymol] 2022; Vol. 669, pp. 91-116. Date of Electronic Publication: 2022 Feb 03.
DOI:	10.1016/bs.mie.2021.12.015
Abstrakt:	The cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster, SAM, and Cbl to carry out remarkable catalytic feats in a large number of biosynthetic pathways. However, despite the abundance of annotated Cbl-dependent radical SAM enzymes, relatively few molecular details exist regarding how these enzymes function. Traditionally, challenges associated with purifying and reconstituting Cbl-dependent radical SAM enzymes have hindered biochemical studies aimed at elucidating the structures and mechanisms of these enzymes. Herein, we describe a bottom-up approach that was used to crystallize OxsB, learn about the overall architecture of a Cbl-dependent radical SAM enzyme, and facilitate mechanistic studies. We report lessons learned from the crystallization of different states of OxsB, including the apo-, selenomethionine (SeMet)-labeled, and fully reconstituted form of OxsB that has a [4Fe-4S] cluster, SAM, and Cbl bound. Further, we suggest that, when appropriate, this bottom-up method can be used to facilitate studies on enzymes in this class for which there are challenges associated with purifying and reconstituting the active enzyme. (Copyright © 2022 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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