Structural and kinetic isotope effect studies of nicotinamidase (Pnc1) from Saccharomyces cerevisiae.
Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD(+) salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from Saccharomyces cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction in which ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogues acting as inhibitors or substrates were examined, revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary (15)N and (13)C kinetic isotope effects (KIEs) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased, indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was indicative of a stronger commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid, is discussed. These results will aid in the design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.
Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States.
Brian C
BC
Smith
Mark A
MA
Anderson
Kelly A
KA
Hoadley
James L
JL
Keck
W Wallace
WW
Cleland
John M
JM
Denu
D000641:Ammonia / Q000737:chemistry; D000818:Animals; D002247:Carbon Isotopes / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002417:Cattle; D018360:Crystallography, X-Ray; D016503:Drug Delivery Systems; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009525:Niacin / Q000037:antagonists & inhibitors / Q000737:chemistry; D009536:Niacinamide / Q000031:analogs & derivatives / Q000737:chemistry; D009535:Nicotinamidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D009587:Nitrogen Isotopes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D002247:Carbon Isotopes; D007656:Ketoglutaric Acids; D009587:Nitrogen Isotopes; D029701:Saccharomyces cerevisiae Proteins; D009536:Niacinamide; D009525:Niacin; D000641:Ammonia; D005969:Glutamate Dehydrogenase; D009535:Nicotinamidase; C474330:PNC1 protein, S cerevisiae