Abstact

Nicotinamidases (PncA) catalyze the hydrolysis of nicotinamide to nicotinic acid, a key step in NAD(+) salvage pathways. In the Lyme disease spirochete Borrelia burgdorferi, the plasmid-encoded gene bbe22 encodes a PncA enzyme that is essential for survival in both mammalian and tick hosts. Previous genetic and biochemical studies demonstrated that translation of B. burgdorferi PncA initiates from a rare non-canonical AUU start codon, resulting in a protein that is 24 residues longer than the sequence currently annotated in major databases. Despite these findings, public resources such as UniProt and KEGG still list a truncated protein beginning at residue 36, which lacks part of the N-terminal region required for enzymatic activity. Here we report the crystal structure of full-length B. burgdorferi PncA determined at 3.2 A resolution. The structure reveals the canonical fold of bacterial nicotinamidases and clear electron density for a ligand in the active site consistent with nicotinic acid, the product of the enzymatic reaction. Structural comparison with homologous PncA enzymes demonstrates conservation of the catalytic architecture, including residues involved in substrate binding and catalysis. Importantly, the experimentally determined structure confirms that the longer N-terminal sequence described previously is required for formation of the correct fold and active-site geometry, whereas the truncated annotation is structurally inconsistent with the observed fold and with AlphaFold predictions. Our results provide the first structural characterization of B. burgdorferi PncA and resolve the long-standing annotation discrepancy for bbe22, validating the correct protein sequence and providing the structural basis for nicotinamidase activity in this essential metabolic enzyme.