Characterization of auxiliary iron-sulfur clusters in a radical S-adenosylmethionine enzyme PqqE from Methylobacterium extorquens AM1.

Abstract

PqqE is a radical S‐adenosyl‐l‐methionine (SAM) enzyme that catalyzes the initial reaction of pyrroloquinoline quinone (PQQ) biosynthesis. PqqE belongs to the SPASM (subtilosin/PQQ/anaerobic sulfatase/mycofactocin maturating enzymes) subfamily of the radical SAM superfamily and contains multiple Fe–S clusters. To characterize the Fe–S clusters in PqqE from Methylobacterium extorquens AM1, Cys residues conserved in the N‐terminal signature motif (CX 3 CX 2C) and the C‐terminal seven‐cysteine motif (CX 9–15 GX 4 CX n CX 2 CX 5 CX 3 CX nC; n = an unspecified number) were individually or simultaneously mutated into Ser. Biochemical and Mössbauer spectral analyses of as‐purified and reconstituted mutant enzymes confirmed the presence of three Fe–S clusters in PqqE: one [4Fe–4S]2+ cluster at the N‐terminal region that is essential for the reductive homolytic cleavage of SAM into methionine and 5′‐deoxyadenosyl radical, and one each [4Fe–4S]2+ and [2Fe–2S]2+ auxiliary clusters in the C‐terminal SPASM domain, which are assumed to serve for electron transfer between the buried active site and the protein surface. The presence of [2Fe–2S]2+ cluster is a novel finding for radical SAM enzyme belonging to the SPASM subfamily. Moreover, we found uncommon ligation of the auxiliary [4Fe–4S]2+ cluster with sulfur atoms of three Cys residues and a carboxyl oxygen atom of a conserved Asp residue.