Mechanistic Insights into Nicotinamidase by CoII EPR Spectroscopy

Abstract

Nicotinamidases catalyze the metal-dependent hydrolysis of nicotinamide in the NAD+ salvage pathway in most nonmammalian organisms. Their essential role in pathogenic bacteria, but not mammals, has made them an attractive target for new antibiotic treatments. Most of the characterized nicotinamidases rely on a ZnII active site for catalysis, which has limited spectroscopic studies of these enzymes. Herein, we employ CoII-substitution as a spectroscopic probe to investigate the mechanism of nicotinamidase from the pathogen Streptococcus pneumoniae (SpPncA). The CoII-substituted enzyme features a spin S = 3/2 EPR signal with resolved hyperfine splitting consistent with a five-coordinate metal center. Analysis of a conserved second-sphere Asp9 residue via site-directed mutagenesis reveals an unexpected role in maintaining the geometry of the metal coordination site through an extended network of electrostatic interactions. Characterization of substrate, intermediate, product, and inhibitor-bound complexes demonstrates that subtle changes in the ligand impact the rhombicity of the EPR spectrum. Leveraging these spectroscopic features, we characterize a catalytically relevant thioester intermediate formed during turnover with nicotinamide for the first time. Together, these results provide new mechanistic insights into nicotinamidase catalysis.