The new epoch of structural insights into radical SAM enzymology.

The Radical SAM (RS) superfamily of enzymes catalyzes a wide array of enzymatic reactions. The majority of these enzymes employ an electron from a reduced [4Fe-4S]+1 cluster to facilitate the reductive cleavage of S-adenosyl-l-methionine, thereby producing a highly reactive 5'-deoxyadenosyl radical (5'-dA⋅) and l-methionine. Typically, RS enzymes use this 5'-dA⋅ to extract a hydrogen atom from the target substrate, starting the cascade of an expansive and impressive variety of chemical transformations. While a great deal of understanding has been gleaned for 5'-dA⋅ formation, because of the chemical diversity within this superfamily, the subsequent chemical transformations have only been fully elucidated in a few examples. In addition, with the advent of new sequencing technology, the size of this family now surpasses 700,000 members, with the number of uncharacterized enzymes and domains also rapidly expanding. In this review, we outline the history of RS enzyme characterization in what we term "epochs" based on advances in technology designed for stably producing these enzymes in an active state. We propose that the state of the field has entered the fourth epoch, which we argue should commence with a protein structure initiative focused solely on RS enzymes to properly tackle this unique superfamily and uncover more novel chemical transformations that likely exist.

Investigation of the Importance of Protein 3D Structure for Assessing Conservation of Lysine Acetylation Sites in Protein Homologs.

Acetylation is a protein post-translational modification (PTM) that can affect a variety of cellular processes. In bacteria, two PTM Nε-acetylation mechanisms have been identified: non-enzymatic/chemical acetylation via acetyl phosphate or acetyl coenzyme A and enzymatic acetylation via protein acetyltransferases. Prior studies have shown that extensive acetylation of Nε-lysine residues of numerous proteins from a variety of bacteria occurs via non-enzymatic acetylation. In Escherichia coli, new Nε-lysine acetyltransferases (KATs) that enzymatically acetylate other proteins have been identified, thus expanding the repertoire of protein substrates that are potentially regulated by acetylation. Therefore, we designed a study to leverage the wealth of structural data in the Protein Data Bank (PDB) to determine: (1) the 3D location of lysine residues on substrate proteins that are acetylated by E. coli KATs, and (2) investigate whether these residues are conserved on 3D structures of their homologs. Five E. coli KAT substrate proteins that were previously identified as being acetylated by YiaC and had 3D structures in the PDB were selected for further analysis: adenylate kinase (Adk), isocitrate dehydrogenase (Icd), catalase HPII (KatE), methionyl-tRNA formyltransferase (Fmt), and a peroxide stress resistance protein (YaaA). We methodically compared over 350 protein structures of these E. coli enzymes and their homologs; to accurately determine lysine residue conservation requires a strategy that incorporates both flexible structural alignments and visual inspection. Moreover, our results revealed discrepancies in conclusions about lysine residue conservation in homologs when examining linear amino acid sequences compared to 3D structures.

Generating enzyme and radical-mediated bisubstrates as tools for investigating Gcn5-related N-acetyltransferases.

Gcn5-related N-acetyltransferases (GNATs) are found in all kingdoms of life and catalyze important acyl transfer reactions in diverse cellular processes. While many 3D structures of GNATs have been determined, most do not contain acceptor substrates in their active sites. To expand upon existing crystallographic strategies for improving acceptor-bound GNAT structures, we synthesized peptide substrate analogs and reacted them with CoA in PA4794 protein crystals. We found two separate mechanisms for bisubstrate formation: (a) a novel X-ray induced radical-mediated alkylation of CoA with an alkene peptide and (b) direct alkylation of CoA with a halogenated peptide. Our approach is widely applicable across the GNAT superfamily and can be used to improve the success rate of obtaining liganded structures of other acyltransferases.