Characterization of nicotinamidases: steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism.
Nicotinamidases are metabolic enzymes that hydrolyze nicotinamide to nicotinic acid. These enzymes are widely distributed across biology, with examples found encoded in the genomes of Mycobacteria, Archaea, Eubacteria, Protozoa, yeast, and invertebrates, but there are none found in mammals. Although recent structural work has improved our understanding of these enzymes, their catalytic mechanism is still not well understood. Recent data show that nicotinamidases are required for the growth and virulence of several pathogenic microbes. The enzymes of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans regulate life span in their respective organisms, consistent with proposed roles in the regulation of NAD(+) metabolism and organismal aging. In this work, the steady state kinetic parameters of nicotinamidase enzymes from C. elegans, Sa. cerevisiae, Streptococcus pneumoniae (a pathogen responsible for human pneumonia), Borrelia burgdorferi (the pathogen that causes Lyme disease), and Plasmodium falciparum (responsible for most human malaria) are reported. Nicotinamidases are generally efficient catalysts with steady state k(cat) values typically exceeding 1 s(-1). The K(m) values for nicotinamide are low and in the range of 2 -110 μM. Nicotinaldehyde was determined to be a potent competitive inhibitor of these enzymes, binding in the low micromolar to low nanomolar range for all nicotinamidases tested. A variety of nicotinaldehyde derivatives were synthesized and evaluated as inhibitors in kinetic assays. Inhibitions are consistent with reaction of the universally conserved catalytic Cys on each enzyme with the aldehyde carbonyl carbon to form a thiohemiacetal complex that is stabilized by a conserved oxyanion hole. The S. pneumoniae nicotinamidase can catalyze exchange of (18)O into the carboxy oxygens of nicotinic acid with H(2)(18)O. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explains nicotinamidase and nicotinic acid (18)O exchange chemistry for the S. pneumoniae enzyme involving key catalytic residues, a catalytic transition metal ion, and the intermediacy of a thioester intermediate.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.
Jarrod B
JB
French
Yana
Y
Cen
Tracy L
TL
Vrablik
Ping
P
Xu
Eleanor
E
Allen
Wendy
W
Hanna-Rose
Anthony A
AA
Sauve
D000447:Aldehydes / Q000737:chemistry / Q000493:pharmacokinetics*; D000595:Amino Acid Sequence; D000818:Animals; D025065:Borrelia burgdorferi / Q000201:enzymology; D017173:Caenorhabditis elegans / Q000201:enzymology; D002384:Catalysis / Q000187:drug effects; D004331:Drosophila melanogaster / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D009535:Nicotinamidase / Q000037:antagonists & inhibitors* / Q000145:classification / Q000493:pharmacokinetics*; D010963:Plasmodium falciparum / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D000447:Aldehydes; C014785:3-pyridinaldehyde; D009535:Nicotinamidase