Inhibition of nicotinamide deamidase from Micrococcus lysodeikticus by analogues of nicotinamide

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

doi:10.1016/0020-711x(74)90027-5

Cited by 10 publications

(7 citation statements)

References 27 publications

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“…Nicotinaldehyde displayed by far the most potent inhibition among the analogs tested with a K i value of 940 nM (Table 1; Figure S1), 10-fold below the K m value of 9.6 µM for nicotinamide. Potent inhibition by nicotinaldehyde was previously observed for nicotinamidases from M. lysodeikticus ( K i = 18 nM) (49), S. cerevisiae ( K i = 1.4 µM) (30), and Mycobacterium tuberculosis ( K i = 290 nM) (33). Benzaldehyde was the weakest inhibitor tested with a K i value of 20.6 mM, similar to the previously observed benzaldehyde inhibition of M. lysodeikticus nicotinamidase ( K i = 1.8 mM) (49).…”

Section: Resultsmentioning

confidence: 74%

“…Benzaldehyde was the weakest inhibitor tested with a K i of 20.6 mM, similar to the previously observed benzaldehyde inhibition of Mi. lysodeikticus nicotinamidase ( K i = 1.8 mM) …”

Section: Resultsmentioning

confidence: 99%

“…The competitive inhibition observed with 3- acetylpyridine [K i = 316 μM (Table 1)] was consistent with several previous reports on nicotinamidases from Mycobacterium phlei, 48 Mi. lysodeikticus (K i = 160 μM), 49 Tortula cremoris (K i = 305 μM), 50 Fleischmann's yeast (K i = 65 μM), 47 Flavobacterium peregrinum, 51 and S. cerevisiae (K i = 46 μM). 30 Nicotinaldehyde displayed by far the most potent inhibition among the analogues tested with a K i value of 940 nM (Table 1 and Figure S1 of the Supporting Information), 10-fold below the K m value of 9.6 μM for nicotinamide.…”

Section: ■ Resultsmentioning

confidence: 99%

“…First, nicotinamide analogue aldehydes and ketones are competitive inhibitors versus nicotinamide. Although these aldehydes and ketones are predicted to form covalent adducts with C167, classical competitive inhibition patterns and no time-dependent loss of activity are expected as the reaction with C167 is completely reversible ,, and infinite concentrations of nicotinamide would preclude binding of the inhibitor to Pnc1. Nicotinaldehyde is a particularly potent inhibitor ( K i value of 940 nM), and the weaker inhibition of 3-acetylpyridine ( K i = 316 μM) is consistent with the greater reactivity of aldehydes versus ketones .…”

Section: Discussionmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 74%

“…Benzaldehyde was the weakest inhibitor tested with a K i of 20.6 mM, similar to the previously observed benzaldehyde inhibition of Mi. lysodeikticus nicotinamidase ( K i = 1.8 mM) …”

Section: Resultsmentioning

confidence: 99%

Section: ■ Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

et al. 2011

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“…Nicotinamidase activity was first reported by Williamson and Hughes in extracts from Lactobacillus arabinosus (13) and this activity was later observed in many other microorganisms (14-19). Nicotinamidases have also been confirmed in plants (20, 21), Caenorhabditis elegans (22), and Drosophila melanogaster (23).…”

mentioning

confidence: 95%

High-Resolution Crystal Structures of Streptococcus pneumoniae Nicotinamidase with Trapped Intermediates Provide Insights into the Catalytic Mechanism and Inhibition by Aldehydes,

et al. 2010

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Nicotinamidases are salvage enzymes that convert nicotinamide to nicotinic acid. These enzymes are essential for the recycling of nicotinamide into NAD+ in most prokaryotes, most single cell and multicellular eukaryotes, but not in mammals. The significance of these enzymes for nicotinamide salvage and for NAD+ homeostasis has increased interest in nicotinamidases as possible antibiotic targets. Nicotinamidases are also regulators of intracellular nicotinamide concentrations, thereby regulating signaling of downstream NAD+ consuming enzymes, such as the NAD+-dependent deacetylases (sirtuins). Here, we report several high resolution crystal structures of the nicotinamidase from Streptococcus pneumoniae (SpNic) in unliganded and ligand-bound forms. The structure of the C136S mutant in complex with nicotinamide provides details about substrate binding while a trapped nicotinoyl-thioester complexed with SpNic reveals the structure of the proposed thioester reaction intermediate. Examination of the active site of SpNic reveals several important features including a metal ion that coordinates the substrate and the catalytically relevant water molecule, and an oxyanion hole which both orients the substrate and offsets the negative charge that builds up during catalysis. Structures of this enzyme with bound nicotinaldehyde inhibitors elucidate the mechanism of inhibition and provide further details about the catalytic mechanism. In addition, we provide a biochemical analysis of the identity and role of the metal ion that orients the ligand in the active site and activates the water molecule responsible for hydrolysis of the substrate. These data provide structural evidence for several proposed reaction intermediates and allow for a more complete understanding of the catalytic mechanism of this enzyme.

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Nicotinamidase

Schomburg¹,

Salzmann²

1991

Enzyme Handbook 4

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Inhibition of nicotinamide deamidase from Micrococcus lysodeikticus by analogues of nicotinamide

Cited by 10 publications

References 27 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

High-Resolution Crystal Structures of Streptococcus pneumoniae Nicotinamidase with Trapped Intermediates Provide Insights into the Catalytic Mechanism and Inhibition by Aldehydes,

Nicotinamidase

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