Dual roles of F 123 in protein homodimerization and inhibitor binding to biotin protein ligase from S taphylococcus aureus

Summary Protein biotinylation is catalysed by biotin protein ligase ( BPL ). The most characterized BPL is from E scherichia coli where it functions as both a biotin ligase and a homodimeric transcriptional repressor. Here we investigated another bifunctional BPL from the clinically important S taphylococcus aureus ( Sa BPL ). Unliganded Sa BPL (apo) exists in a dimer‐monomer equilibrium at low micromolar concentrations – a stark contrast to E . coli   BPL ( Ec BPL ) that is monomeric under the same conditions. EMSA and SAXS analysis demonstrated that dimeric apo Sa BPL adopted a conformation that was competent to bind DNA and necessary for it to function as a transcription factor. The Sa BPL dimer‐monomer dissociation constant was 5.8‐fold tighter when binding the inhibitor biotin acetylene, but unchanged with biotin. F 123, located in the dimer interface, was critical for homodimerization. Inhibition studies together with surface plasmon resonance analyses revealed a strong correlation between inhibitor potency and slow dissociation kinetics. A 24‐fold difference in K i values for these two enzymes was explained by differences in enzyme:inhibitor dissociation rates. Substitution of F 123 in Sa BPL and its equivalent in Ec BPL altered both inhibitor potency and dissociation. Hence, F 123 in Sa BPL has novel roles in both protein dimerization and ligand‐binding that have not been reported in Ec BPL .