Characterization of nicotinamidases: Steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism

Jarrod B. French, Yana Cen, Tracy L. Vrablik, Ping Xu, Eleanor Allen, Wendy Hanna-Rose, Anthony A. Sauve

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

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 kcat values typically exceeding 1 s -1. The Km 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 18O into the carboxy oxygens of nicotinic acid with H218O. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explains nicotinamidase and nicotinic acid 18O exchange chemistry for the S. pneumoniae enzyme involving key catalytic residues, a catalytic transition metal ion, and the intermediacy of a thioester intermediate.

Original languageEnglish (US)
Pages (from-to)10421-10439
Number of pages19
JournalBiochemistry
Volume49
Issue number49
DOIs
StatePublished - Dec 14 2010

Fingerprint

Nicotinamidase
Kinetic parameters
Enzymes
Niacin
Streptococcus pneumoniae
Yeast
Niacinamide
Caenorhabditis elegans
Pathogens
Saccharomyces cerevisiae
Protozoa
Borrelia burgdorferi
Kinetics
Mammals
Lyme Disease
Archaea
Enzyme Inhibitors
Invertebrates
Plasmodium falciparum
Mycobacterium

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

French, Jarrod B. ; Cen, Yana ; Vrablik, Tracy L. ; Xu, Ping ; Allen, Eleanor ; Hanna-Rose, Wendy ; Sauve, Anthony A. / Characterization of nicotinamidases : Steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism. In: Biochemistry. 2010 ; Vol. 49, No. 49. pp. 10421-10439.
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abstract = "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 kcat values typically exceeding 1 s -1. The Km 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 18O into the carboxy oxygens of nicotinic acid with H218O. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explains nicotinamidase and nicotinic acid 18O exchange chemistry for the S. pneumoniae enzyme involving key catalytic residues, a catalytic transition metal ion, and the intermediacy of a thioester intermediate.",
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Characterization of nicotinamidases : Steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism. / French, Jarrod B.; Cen, Yana; Vrablik, Tracy L.; Xu, Ping; Allen, Eleanor; Hanna-Rose, Wendy; Sauve, Anthony A.

In: Biochemistry, Vol. 49, No. 49, 14.12.2010, p. 10421-10439.

Research output: Contribution to journalArticle

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T2 - Steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism

AU - French, Jarrod B.

AU - Cen, Yana

AU - Vrablik, Tracy L.

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AU - Allen, Eleanor

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AU - Sauve, Anthony A.

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