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

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes are crucial for various biological reactions, primarily methylations.
• Canonical Cbl-binding domains in these enzymes typically feature an N-terminal Rossmann fold.
• Methanogenesis marker protein 10 (Mmp10) utilizes Cbl for methylation but possesses a divergent C-terminal Cbl-binding domain.

Purpose of the Study:

• To investigate the Domain of Unknown Function 512 (DUF512) as a novel Cbl-binding domain in radical SAM enzymes.
• To characterize the structure and Cbl-binding capabilities of DUF512-containing proteins.

Main Methods:

• Bioinformatic analysis to identify DUF512 domains in radical SAM enzymes.
• Overexpression and purification of four selected DUF512-containing proteins.
• Cbl-binding assays and X-ray crystallography to determine protein structures.

Main Results:

• Four DUF512-containing proteins were confirmed to bind Cbl.
• X-ray crystal structures revealed Cbl-binding domains within DUF512.
• The structure from *Clostridium sporogenes* is the first radical SAM enzyme structure with a PDZ domain and an unprecedented β3α4 core; the *Pyrococcus furiosus* structure shows an uncommon (βα)5 core.

Conclusions:

• DUF512 represents a novel class of Cbl-binding domains in radical SAM enzymes.
• These findings expand the known structural diversity of Cbl-dependent radical SAM enzymes, particularly in bacteria.
• The unique structural cores identified provide new insights into the catalytic mechanisms of these enzymes.