Peptide Selenocysteine Substitutions Reveal Direct Substrate-Enzyme Interactions at Auxiliary Clusters in Radical S-Adenosyl-l-methionine Maturases.
J Am Chem Soc
; 145(18): 10167-10177, 2023 05 10.
Article
em En
| MEDLINE
| ID: mdl-37104670
ABSTRACT
Radical S-adenosyl-l-methionine (SAM) enzymes leverage the properties of one or more iron- and sulfide-containing metallocenters to catalyze complex and radical-mediated transformations. By far the most populous superfamily of radical SAM enzymes are those that, in addition to a 4Fe-4S cluster that binds and activates the SAM cofactor, also bind one or more additional auxiliary clusters (ACs) of largely unknown catalytic significance. In this report we examine the role of ACs in two RS enzymes, PapB and Tte1186, that catalyze formation of thioether cross-links in ribosomally synthesized and post-translationally modified peptides (RiPPs). Both enzymes catalyze a sulfur-to-carbon cross-link in a reaction that entails H atom transfer from an unactivated C-H to initiate catalysis, followed by formation of a C-S bond to yield the thioether. We show that both enzymes tolerate substitution of SeCys instead of Cys at the cross-linking site, allowing the systems to be subjected to Se K-edge X-ray spectroscopy. The EXAFS data show a direct interaction with the Fe of one of the ACs in the Michaelis complex, which is replaced with a Se-C interaction under reducing conditions that lead to the product complex. Site-directed deletion of the clusters in Tte1186 provide evidence for the identity of the AC. The implications of these observations in the context of the mechanism of these thioether cross-linking enzymes are discussed.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Selenocisteína
Idioma:
En
Revista:
J Am Chem Soc
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos