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

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 ( CX₃ CX₂C) and the C-terminal seven-cysteine motif ( CX_9-15 GX₄ CX _n CX₂ CX₅ CX₃ 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. [ABSTRACT FROM AUTHOR]