Ornithine racemase uses a catalytic cysteine

ABSTRACT

Salmonella enterica serover typhimurium expresses an ornithine racemase (OrnR), located in a small operon containing amino acid transporter homologues and D-ornithine/d-lysine decarboxylase. This OrnR is part of a clade that has high identity (>75 %) with other sequences in enterobacteria, but is distant (∼40 % identity) from another clade of OrnR found in anaerobic Firmicutes and actinobacteria. OrnR is a member of the pyridoxal-5'-phosphate (PLP)-dependent alanine racemase (AlaR) superfamily (Fold III), but sequence alignments show that it lacks the catalytic Tyr acid/base of the other racemases. However, the sequences show a conserved cysteine. OrnR shows hyperbolic kinetics in the L → D direction, but exhibits sigmoid kinetics in the D → L direction, with n = 1.8. The structure of OrnR predicted by AlphaFold3 was complexed with PLP-DL-ornithine in silico, minimized, and subjected to molecular dynamics simulation. The resulting structure shows that Cys-164 is in the active site, about 4 Å from the Cα of the external aldimine of L-ornithine, while the NZ of Lys-36 is located about 4 Å below Cα on the D-face. The C164A mutant enzyme has no measurable racemization activity, and does not exchange the α-H of L- or D-ornithine in D2O, consistent with the function of Cys-164 as an acid/base catalyst. These data are consistent with a concerted mechanism for the racemization. Gel filtration of OrnR shows a mixture of dimeric and tetrameric assemblies.