Kinetic studies of nicotinamide deamidase from Micrococcus lysodeikticus

Gadd, R. E. A.; Johnson, Willard J.

doi:10.1016/0020-711x(74)90136-0

Cited by 12 publications

(12 citation statements)

References 26 publications

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“…Double-reciprocal analysis of nicotinamidase inhibition by ammonium ion using a previously published HPLC assay (45) exhibited non-competitive inhibition patterns (data not shown). However, greater than 10 mM ammonium chloride was required for inhibition, consistent with several previous reports on nicotinamidases from M. lysodeikticus and yeast (46, 47). Therefore, it was unclear if the ammonium ion added was acting as a product inhibitor or as a denaturant as non-competitive inhibition patterns would be predicted in either case.…”

Section: Resultssupporting

confidence: 91%

“…Double-reciprocal analysis of nicotinamidase inhibition by nicotinic acid and pyrazinoic acid displayed competitive inhibition patterns with K i values of 120 µM and 6.7 mM, respectively (Table 1; Figure S1). The competitive inhibition displayed by nicotinic acid is consistent with that shown by Gadd et al for a nicotinamidase from Micrococcus lysodeikticus (46) but in contrast to French et al who observed no inhibition with nicotinic acid for S. cerevisiae Pnc1 (30). Double-reciprocal analysis of nicotinamidase inhibition by ammonium ion using a previously published HPLC assay (45) exhibited non-competitive inhibition patterns (data not shown).…”

Section: Resultssupporting

confidence: 89%

“…The K m value of 157 µM determined for pyrazinamide was similar to previously determined values for nicotinamidases from S. cerevisiae ( K m = 200 µM) (2) and M. tuberculosis ( K m = 300 µM) (33). The K m value of 61 µM determined for 5-methylnicotinamide was 5-fold lower than a previously determined value of 360 µM for a nicotinamidase from M. lysodeikticus (46). Perturbation of the pyridine nitrogen of nicotinamide by methylation (1-methylnicotinamide) or substitution with carbon (benzamide) resulted in 8400- and 200-fold decreased k cat / K m values compared to nicotinamide.…”

Section: Resultscontrasting

confidence: 72%

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Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

et al. 2011

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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 S. cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction where ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogs 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 15N and 13C kinetic isotope effects (KIE) 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 observed indicative of a higher 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 is discussed involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid. These results will aid design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.

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Section: Resultssupporting

confidence: 91%

Section: Resultssupporting

confidence: 89%

Section: Resultscontrasting

confidence: 72%

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Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

et al. 2011

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“…Although nicotinamide was widespread in the environment, information of microbial degradation of nicotinamide is quite limited. The study of microbial transformation of nicotinamide mainly focus on the first deamination step 12,13 . In the deamination step, nicotinamide was transformed to nicotinic acid by nicotinamidase releasing NH 4 + 13 .…”

Section: Introductionmentioning

confidence: 99%

“…The study of microbial transformation of nicotinamide mainly focus on the first deamination step 12,13 . In the deamination step, nicotinamide was transformed to nicotinic acid by nicotinamidase releasing NH 4 + 13 . This reaction is not only involved in NAD + biosynthesis, but in many other important physiological processes.…”

Section: Introductionmentioning

confidence: 99%

Microbial Degradation of Nicotinamide by a Strain Alcaligenes sp. P156

Zhao

et al. 2019

Sci Rep

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A novel Alcaligenes sp. strain P156, which can utilize nicotinamide as its sole source of carbon, nitrogen and energy, was enriched and isolated from soil in a solid waste treatment plant. Aerobic growth and degradation with nicotinamide were characterized. Seven nicotinamide degradation-related genes were obtained by sequence alignment from the genome sequence of strain P156. Four genes, designated naaA , naaD , naaE and naaF , were cloned and heterologously expressed. Nicotinamide degradation is initiated by deamination to form nicotinic acid catalyzed by the nicotinamidase NaaA, which shares highest amino acid sequence identity (27.2%) with nicotinamidase from Arabidopsis thaliana . Nicotinic acid is converted to 6-hydroxynicotinic acid, which is further oxidized to 2,5-dihydroxypyridine (2,5-DHP). 2,5-DHP is then transformed to a ring-cleavage product, N -formylmaleamic acid, by an Fe 2+ dependent dioxygenase NaaD. N -formylmaleamic acid is transformed to fumaric acid through maleamic acid and maleic acid by NaaE and NaaF, respectively. To our knowledge, this is the first report of the complete microbial degradation of nicotinamide in bacteria. Nicotinamide is considered as a model compound for the study of microbial degradation of pyridinic compounds, and the nicotinamide degrading related genes in strain P156 were distributed differently from the reported similar gene clusters. Therefore, this study contribute to the knowledge on the degradation of pyridinic compounds.

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Hydrolases

Spector

1982

Covalent Catalysis by Enzymes

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Kinetic studies of nicotinamide deamidase from Micrococcus lysodeikticus

Cited by 12 publications

References 26 publications

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

Microbial Degradation of Nicotinamide by a Strain Alcaligenes sp. P156

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