<i>Pseudomonas aeruginosa</i> aliphatic amidase is related to the nitrilase/cyanide hydratase enzyme family and Cys<sup>166</sup> is predicted to be the active site nucleophile of the catalytic mechanism

Novo, Carlos; Tata, Renée; Clemente, A.; Brown, Paul

doi:10.1016/0014-5793(95)00585-w

Cited by 58 publications

(39 citation statements)

References 43 publications

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“…The CN_hydrolase domain, with a cysteine residue essential for nitrilase activity (24,25), is shared with enzymes belonging to the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia (Fig. 2C) (26,27). Because B. subtilis synthetase, which lacks the CN_hydro-lase domain ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Molecular Identification of Human Glutamine- and Ammonia-dependent NAD Synthetases

Hara

Yamada

Terashima

et al. 2003

Journal of Biological Chemistry

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NAD synthetase catalyzes the final step in the biosynthesis of NAD. In the present study, we obtained cDNAs for two types of human NAD synthetase (referred as NADsyn1 and NADsyn2). Structural analysis revealed in both NADsyn1 and NADsyn2 a domain required for NAD synthesis from ammonia and in only NADsyn1 an additional carbon-nitrogen hydrolase domain shared with enzymes of the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia. Consistent with the domain structures, biochemical assays indicated (i) that both NADsyn1 and NADsyn2 have NAD synthetase activity, (ii) that NADsyn1 uses glutamine as well as ammonia as an amide donor, whereas NADsyn2 catalyzes only ammoniadependent NAD synthesis, and (iii) that mutant NADsyn1 in which Cys-175 corresponding to the catalytic cysteine residue in nitrilases was replaced with Ser does not use glutamine. Kinetic studies suggested that glutamine and ammonia serve as physiological amide donors for NADsyn1 and NADsyn2, respectively. Both synthetases exerted catalytic activity in a multimeric form. In the mouse, NADsyn1 was seen to be abundantly expressed in the small intestine, liver, kidney, and testis but very weakly in the skeletal muscle and heart. In contrast, expression of NADsyn2 was observed in all tissues tested. Therefore, we conclude that humans have two types of NAD synthetase exhibiting different amide donor specificity and tissue distributions. The ammoniadependent synthetase has not been found in eucaryotes until this study. Our results also indicate that the carbon-nitrogen hydrolase domain is the functional domain of NAD synthetase to make use of glutamine as an amide donor in NAD synthesis. Thus, glutamine-dependent NAD synthetase may be classified as a possible glutamine amidase in the nitrilase family. Our molecular identification of NAD synthetases may prove useful to learn more of mechanisms regulating cellular NAD metabolism.The coenzyme NAD has a role in the majority of metabolic redox reactions and represents an essential component of metabolic pathways in all living cells. In a number of signaling pathways, NAD also serves as a precursor of potent calciummobilizing agents such as cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (1) and serves as a substrate for post-translational modifications of protein, mono-(2-4) and poly(ADP-ribosyl)ations (5). Depletion of cellular NAD by poly-(ADP-ribosyl)transferase activation in response to DNA damage results in cell death (6). Increased NAD synthesis has been shown to extend life span in yeast (7) and in Caenorhabditis elegans (8) via activation of an NAD-dependent histone deacetylase, silent information regulator 2 (Sir2) (9). The cellular level of NAD may modulate the sensitivity of cells to apoptotic responses through deacetylation of the p53 tumor suppressor by a human homologue of Sir2 (10). Recent publications have demonstrated that fluctuation of the NAD level in cells seems to have significant impact on their physiology. Despite the...

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Section: Resultsmentioning

confidence: 99%

Molecular Identification of Human Glutamine- and Ammonia-dependent NAD Synthetases

Hara

Yamada

Terashima

et al. 2003

Journal of Biological Chemistry

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“…However, PCR amplification detected an amiE gene in all H. pylori strains (45 clinical isolates) tested and, thus, the acquisition of the gene predates their common ancestor. Bork and Koonin (1994) and Novo et al (1995) observed that the P. aeruginosa and Rhodococcus sp. R312 aliphatic amidases are structurally related to a family of carbon-nitrogen hydrolases comprising nitrilases, cyanide hydratases and ␤-alanine synthase.…”

Section: Discussionmentioning

confidence: 99%

“…The sequence similarities and the involvement of all these enzymes in the reduction of organic nitrogen compounds and in ammonia production suggests a common catalytic mechanism. The several conserved motifs characterized (Bork and Koonin, 1994;Novo et al, 1995) are found in the three sequenced aliphatic amidases, including the H. pylori amidase. By analogy with the nitrilases (Bork and Koonin, 1994;Novo et al, 1995), a region containing a strictly conserved cysteine residue (C-165 in the H. pylori amidase, underlined in Fig.…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of an aliphatic amidase in Helicobacter pylori

Skouloubris

Labigne

Reuse

1997

Molecular Microbiology

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SummaryWe report, for the first time, the presence in Helicobacter pylori of an aliphatic amidase that, like urease, contributes to ammonia production. Aliphatic amidases are cytoplasmic acylamide amidohydrolases (EC 3.5.1.4) hydrolysing short-chain aliphatic amides to produce ammonia and the corresponding organic acid. The finding of an aliphatic amidase in H. pylori was unexpected as this enzyme has only previously been described in bacteria of environmental (soil or water) origin. The H. pylori amidase gene amiE (1017 bp) was sequenced, and the deduced amino acid sequence of AmiE (37 746 Da) is very similar (75% identity) to the other two sequenced aliphatic amidases, one from Pseudomonas aeruginosa and one from Rhodococcus sp. R312. Amidase activity was measured as the release of ammonia by sonicated crude extracts from H. pylori strains and from recombinant Escherichia coli strains overproducing the H. pylori amidase. The substrate specificity was analysed with crude extracts from H. pylori cells grown in vitro; the best substrates were propionamide, acrylamide and acetamide. Polymerase chain reaction (PCR) amplification of an internal amiE sequence was obtained with each of 45 different H. pylori clinical isolates, suggesting that amidase is common to all H. pylori strains. A H. pylori mutant (N6-836) carrying an interrupted amiE gene was constructed by allelic exchange. No amidase activity could be detected in N6-836. In a N6-urease negative mutant, amidase activity was two-to threefold higher than in the parental strain N6. Crude extracts of strain N6 slowly hydrolysed formamide. This activity was affected in neither the amidase negative strain (N6-836) nor a double mutant strain deficient in both amidase and urease activities, suggesting the presence of an independent discrete formamidase in H. pylori. The existence of an aliphatic amidase, a correlation between the urease and amidase activities and the possible presence of a formamidase indicates that H. pylori has a large range of possibilities for intracellular ammonia production.

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“…This aliphatic amidase is classified as one group belonging to the C-N hydrolase superfamily (36,37). This superfamily included nitrilases that convert the cyano group of a nitrile into a carboxylic acid (38,39) and cyanide hydratases that convert a cyano group of a cyanide into an amide (40,41).…”

Section: Fig 2 Sds͞page Gelmentioning

confidence: 99%

Identification of active sites in amidase: Evolutionary relationship between amide bond- and peptide bond-cleaving enzymes

Kobayashi

Fujiwara

Goda

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

117

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Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of sitedirected mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 3 Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 3 Asn substitution as well as Ser195 3 Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH 2 ) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.

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Pseudomonas aeruginosa aliphatic amidase is related to the nitrilase/cyanide hydratase enzyme family and Cys¹⁶⁶ is predicted to be the active site nucleophile of the catalytic mechanism

Cited by 58 publications

References 43 publications

Molecular Identification of Human Glutamine- and Ammonia-dependent NAD Synthetases

Molecular Identification of Human Glutamine- and Ammonia-dependent NAD Synthetases

Identification and characterization of an aliphatic amidase in Helicobacter pylori

Identification of active sites in amidase: Evolutionary relationship between amide bond- and peptide bond-cleaving enzymes

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Pseudomonas aeruginosa aliphatic amidase is related to the nitrilase/cyanide hydratase enzyme family and Cys166 is predicted to be the active site nucleophile of the catalytic mechanism

Cited by 58 publications

References 43 publications

Molecular Identification of Human Glutamine- and Ammonia-dependent NAD Synthetases

Molecular Identification of Human Glutamine- and Ammonia-dependent NAD Synthetases

Identification and characterization of an aliphatic amidase in Helicobacter pylori

Identification of active sites in amidase: Evolutionary relationship between amide bond- and peptide bond-cleaving enzymes

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Pseudomonas aeruginosa aliphatic amidase is related to the nitrilase/cyanide hydratase enzyme family and Cys¹⁶⁶ is predicted to be the active site nucleophile of the catalytic mechanism