Structural\nEvidence for DUF512 as a Radical <i>S</i>‑Adenosylmethionine\nCobalamin-Binding Domain

Cobalamin (Cbl)-dependent\nradical <i>S</i>-adenosylmethionine\n(SAM) enzymes constitute a large subclass of radical SAM (RS) enzymes\nthat use Cbl to catalyze various types of reactions, the most common\nof which are methylations. Most Cbl-dependent RS enzymes contain an\nN-terminal Rossmann fold that aids Cbl binding. Recently, it has been\ndemonstrated that the methanogenesis marker protein 10 (Mmp10) requires\nCbl to methylate an arginine residue in the α-subunit of methyl\ncoenzyme M reductase. However, Mmp10 contains a Cbl-binding domain\nin the C-terminal region of its primary structure that does not share\nsignificant sequence similarity with canonical RS Cbl-binding domains.\nBioinformatic analysis of Mmp10 identified DUF512 (Domain of Unknown\nFunction 512) as a potential Cbl-binding domain in RS enzymes. In\nthis paper, four randomly selected DUF512-containing proteins from\nvarious organisms were overexpressed, purified, and shown to bind\nCbl. X-ray crystal structures of DUF512-containing proteins from Clostridium sporogenes and Pyrococcus\nfuriosus were determined, confirming their C-terminal\nCbl-binding domains. The structure of the DUF512-containing protein\nfrom C. sporogenes is the first of\nan RS enzyme containing a PDZ domain. Its RS domain has an unprecedented\nβ<sub>3</sub>α<sub>4</sub> core, whereas most RS enzymes\nadopt a (βα)<sub>6</sub> core. The DUF512-containing protein\nfrom P. furiosus has no PDZ domain,\nbut its RS domain also has an uncommon (βα)<sub>5</sub> core.