Not all 5'-deoxyadenosines are created equal: Tracing the provenance of 5'-deoxyadenosine formed by the radical S-adenosyl-L-methionine enzyme 7-carboxy-7-deazaguanine synthase.

Members of the radical S-adenosyl-L-methionine (rSAM) enzyme superfamily cleave SAM to generate the highly reactive 5'-deoxyadenosyl radical (dAdo•), where dAdo• initiates the reaction by an H-atom transfer from the substrate to form 5'-deoxyadenosine (dAdo) in nearly every member of the superfamily. However, in all rSAM enzymes, SAM also undergoes reductive cleavage to form dAdo in a reaction that is apparently uncoupled from the formation of the product. Herein, we examine the dAdo that is formed under catalytic conditions with the rSAM enzyme 7-carboxy-7-deazaguanine synthase (QueE), which catalyzes the radical-mediated transformation of 6-carboxy-5,6,7,8-tetrahydropterin (CPH) to 7-carboxy-7-deazaguanine (CDG).

The binuclear copper state of peptidylglycine monooxygenase visualized through a selenium-substituted peptidyl-homocysteine complex.

Bioactive peptides generally require post-translational processing to convert them to their fully active forms. Peptidylglycine monooxygenase (PHM) is a copper-dependent enzyme that catalyzes C-alpha hydroxylation of a glycine-extended pro-peptide, a critical post-translational step in peptide amidation. A canonical mechanism based on experimental and theoretical considerations proposes that molecular oxygen reacts at the mononuclear CuM-center to form a reactive Cu(II)-superoxo intermediate capable of H-atom abstraction from the peptidyl substrate, followed by long range ET from the CuH center positioned 11 Å away across a solvent-filled cleft.

Capturing the Binuclear Copper State of Peptidylglycine Monooxygenase Using a Peptidyl-Homocysteine Lure.

Peptidylglycine monooxygenase is a copper-dependent enzyme that catalyzes C-alpha hydroxylation of glycine extended pro-peptides, a critical post-translational step in peptide hormone processing. The canonical mechanism posits that dioxygen binds at the mononuclear M-center to generate a Cu(II)-superoxo species capable of H atom abstraction from the peptidyl substrate, followed by long-range electron tunneling from the CuH center. Recent crystallographic and biochemical data have challenged this mechanism, suggesting instead that an "open-to-closed" transition brings the copper centers closer, allowing reactivity within a binuclear intermediate.

A Promiscuous rSAM Enzyme Enables Diverse Peptide Cross-linking.

Ribosomally produced and post-translationally modified polypeptides (RiPPs) are a diverse group of natural products that are processed by a variety of enzymes to their biologically relevant forms. PapB is a member of the radical -adenosyl-l-methionine (rSAM) superfamily that introduces thioether cross-links between Cys and Asp residues in the PapA RiPP. We report that PapB has high tolerance for variations in the peptide substrate.

Insights into the Mechanism of Installation of 5-Carboxymethylaminomethyl Uridine Hypermodification by tRNA-Modifying Enzymes MnmE and MnmG.

The evolutionarily conserved bacterial proteins MnmE and MnmG (and their homologues in Eukarya) install a 5-carboxymethylaminomethyl (cmnm) or a 5-taurinomethyl (τm) group onto wobble uridines of several tRNA species. The MnmE binds guanosine-5'-triphosphate (GTP) and methylenetetrahydrofolate (CHTHF), while MnmG binds flavin adenine dinucleotide (FAD) and a reduced nicotinamide adenine dinucleotide (NADH). Together with glycine, MnmEG catalyzes the installation of cmnm in a reaction that also requires hydrolysis of GTP.