Structural Evidence for DUF512 as a Radical S-Adenosylmethionine Cobalamin-Binding Domain

Abstract

Cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes constitute a large subclass of radical SAM (RS) enzymes that use Cbl to catalyze various types of reactions, the most common of which are methylations. Most Cbl-dependent RS enzymes contain an N-terminal Rossmann fold that aids Cbl binding. Recently, it has been demonstrated that the methanogenesis marker protein 10 (Mmp10) requires Cbl to methylate an arginine residue in the α-subunit of methyl coenzyme M reductase. However, Mmp10 contains a Cbl-binding domain in the C-terminal region of its primary structure that does not share significant sequence similarity with canonical RS Cbl-binding domains. Bioinformatic analysis of Mmp10 identified DUF512 (Domain of Unknown Function 512) as a potential Cbl-binding domain in RS enzymes. In this paper, four randomly selected DUF512-containing proteins from various organisms were overexpressed, purified, and shown to bind Cbl. X-ray crystal structures of DUF512-containing proteins from Clostridium sporogenes and Pyrococcus furiosus were determined, confirming their C-terminal Cbl-binding domains. The structure of the DUF512-containing protein from C. sporogenes is the first of an RS enzyme containing a PDZ domain. Its RS domain has an unprecedented β₃α₄ core, whereas most RS enzymes adopt a (βα)₆ core. The DUF512-containing protein from P. furiosus has no PDZ domain, but its RS domain also has an uncommon (βα)₅ core.