Ornithine racemase uses a catalytic cysteine

Area of Science:

• Enzymology
• Protein Structure
• Microbial Metabolism

Background:

• Salmonella typhimurium utilizes ornithine racemase (OrnR) in a specialized operon.
• OrnR belongs to the pyridoxal-5'-phosphate (PLP)-dependent alanine racemase superfamily.
• Distinct clades of OrnR exist in different bacterial phyla.

Purpose of the Study:

• To elucidate the catalytic mechanism and structural basis of Salmonella typhimurium ornithine racemase (OrnR).
• To investigate the role of conserved residues, particularly cysteine, in OrnR activity.
• To characterize the kinetic properties and oligomeric state of OrnR.

Main Methods:

• Bioinformatic analysis of OrnR sequence identity across bacterial groups.
• In silico structural prediction using AlphaFold3, molecular dynamics simulations.
• Enzymatic assays including kinetic analysis and site-directed mutagenesis (C164A mutant).
• Gel filtration to determine protein oligomeric state.

Main Results:

• OrnR exhibits distinct kinetic profiles for L- to D- and D- to L-ornithine conversion.
• In silico structural analysis identified Cys-164 and Lys-36 in proximity to the ornithine substrate.
• The C164A mutant demonstrated a complete loss of racemization activity, implicating Cys-164 as the catalytic acid/base.
• OrnR exists as a mixture of dimeric and tetrameric forms.

Conclusions:

• Cys-164 acts as the catalytic acid/base residue in a concerted mechanism for ornithine racemization.
• The findings provide insights into the unique catalytic strategy of OrnR compared to other racemases.
• OrnR's oligomeric state may influence its enzymatic function.