Publications

• Role of bicarbonate/CO2 in the inhibition of Escherichia coli growth by cyanate.

  Kozliak E.I., Fuchs J.A., Guilloton M.B., Anderson P.M.

  Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give two CO2 molecules. The gene for cyanase is part of the cyn operon, which includes cynT and cynS, encoding carbonic anhydrase and cyanase, respectively. Carbonic anhydrase functions to ... >> More

  Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give two CO2 molecules. The gene for cyanase is part of the cyn operon, which includes cynT and cynS, encoding carbonic anhydrase and cyanase, respectively. Carbonic anhydrase functions to prevent depletion of cellular bicarbonate during cyanate decomposition (the product CO2 can diffuse out of the cell faster than noncatalyzed hydration back to bicarbonate). Addition of cyanate to the culture medium of a delta cynT mutant strain of E. coli (having a nonfunctional carbonic anhydrase) results in depletion of cellular bicarbonate, which leads to inhibition of growth and an inability to catalyze cyanate degradation. These effects can be overcome by aeration with a higher partial CO2 pressure (M. B. Guilloton, A. F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P. M. Anderson, and J. A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). The question considered here is why depletion of bicarbonate/CO2 due to the action of cyanase on cyanate in a delta cynT strain has such an inhibitory effect. Growth of wild-type E. coli in minimal medium under conditions of limited CO2 was severely inhibited, and this inhibition could be overcome by adding certain Krebs cycle intermediates, indicating that one consequence of limiting CO2 is inhibition of carboxylation reactions. However, supplementation of the growth medium with metabolites whose syntheses are known to depend on a carboxylation reaction was not effective in overcoming inhibition related to the bicarbonate deficiency induced in the delta cynT strain by addition of cyanate. Similar results were obtained with a deltacyn strain (since cyanase is absent, this strain does not develop a bicarbonate deficiency when cyanate is added); however, as with the deltacynT strain, a higher partial CO(2) pressure in the aerating gas or expression of carbonic anhydrase activity (which contributes to a higher intercellular concentration of bicarbonate/CO(2)) significantly reduced inhibition of growth. There appears to be competition between cyanate and bicarbonate/CO(2) at some unknown but very important site such that cyanate binding inhibits growth. These results suggest that bicarbonate/CO(2) plays a significant role in the growth of E. coli other than simply as a substrate for carboxylation reactions and that strains with mutations in the cyn operon provide a unique model system for studying aspects of the metabolism of bicarbonate/CO(2) and its regulation in bacteria. << Less

  J Bacteriol 177:3213-3219(1995) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Reaction of the N-terminal methionine residues in cyanase with diethylpyrocarbonate.

  Anderson P.M., Korte J.J., Holcomb T.A.

  Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The enzyme is a decamer of identical subunits (M(r) = 17,000). Previous studies have shown that modification of either the single cysteine residue or the s ... >> More

  Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The enzyme is a decamer of identical subunits (M(r) = 17,000). Previous studies have shown that modification of either the single cysteine residue or the single histidine residue in each subunit gives an active decameric derivative that dissociates reversibly to inactive dimer derivative, indicating that decameric structure is required for activity and that the SH and imidazole groups are not required for catalytic activity [Anderson, P. M., Korte, J. J., Holcomb, T. A., Cho, Y.-G., Son, C.-M., & Sung, Y.-C. (1994) J. Biol. Chem. 269, 15036-15045]. Here the effects of reaction of the reagent diethylpyrocarbonate (DEPC) with cyanase or mutant cyanases are reported. DEPC reacts stoichiometrically with the histidine residue and at one additional site in each subunit when the enzyme is in the inactive dimer form, preventing reactivation. DEPC reacts stoichiometrically (with the same result on reactivation) at only one site per subunit with the inactive dimer form of cyanase mutants in which the single histidine residue has been replaced by one of several different amino acids by site-directed mutagenesis; the site of the reaction was identified as the amino group of the N-terminal methionine. DEPC does not react with the histidine residue of the active decameric form of wild-type cyanase and does not affect activity of the active decameric form of wild-type or mutant cyanases. Reaction with the N-terminal amino group of methionine apparently prevents reactivation of the mutant enzymes by blocking association to decamer.(ABSTRACT TRUNCATED AT 250 WORDS) << Less

  Biochemistry 33:14121-14125(1994) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Bicarbonate is a recycling substrate for cyanase.

  Johnson W.V., Anderson P.M.

  Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as su ... >> More

  Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3-serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN-in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3-is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3-is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate. << Less

  J Biol Chem 262:9021-9025(1987) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and properties of the inducible enzyme cyanase.

  Anderson P.M.

  Cyanase (cyanate hydrolase EC 3.5.5.3) has been purified 270-fold to a high state of purity from Escherichia coli B. The native enzyme has a molecular weight of approximately 150 000 as estimated by sucrose density gradient centrifugation and gel-filtration chromatography on Bio-Gel P-300. The enz ... >> More

  Cyanase (cyanate hydrolase EC 3.5.5.3) has been purified 270-fold to a high state of purity from Escherichia coli B. The native enzyme has a molecular weight of approximately 150 000 as estimated by sucrose density gradient centrifugation and gel-filtration chromatography on Bio-Gel P-300. The enzyme is an oligomer composed of apparently identical subunits which have a molecular weight of approximately 15 000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analyses showed that the enzyme contains no tryptophan and a single histidine residue, based on a subunit molecular weight of 14 661. Catalytic hydrolysis of cyanate was found to be dependent on the patience of bicarbonate and to be affected by ionic strength. The concentration of bicarbonate required to give half-maximal activity in the presence of 2 mM potassium cyanate was 0.1 mM. The apparent Km for cyanate in the presence of 3 mM bicarbonate is 0.6 mM. The initial product of the reaction is carbamate (or a related, unstable compound and/or carbamate precursor) which subsequently decomposes to ammonia and bicarbonate. << Less

  Biochemistry 19:2882-2888(1980) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The synthesis of the induced enzyme, ''cyanase'', in E. coli.

  TAUSSIG A.

  Biochim Biophys Acta 44:510-519(1960) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site.

  Walsh M.A., Otwinowski Z., Perrakis A., Anderson P.M., Joachimiak A.

  <h4>Background</h4>Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide. In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate. The enzyme is functionally active as ... >> More

  <h4>Background</h4>Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide. In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate. The enzyme is functionally active as a homodecamer of 17 kDa subunits, and displays half-site binding of substrates or substrate analogs. The enzyme shows no significant amino acid sequence homology with other proteins.<h4>Results</h4>We have determined the crystal structure of cyanase at 1.65 A resolution using the multiwavelength anomalous diffraction (MAD) method. Cyanase crystals are triclinic and contain one homodecamer in the asymmetric unit. Selenomethionine-labeled protein offers 40 selenium atoms for use in phasing. Structures of cyanase with bound chloride or oxalate anions, inhibitors of the enzyme, allowed identification of the active site.<h4>Conclusions</h4>The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding alpha-helix bundle motif. The C-terminal domain has an 'open fold' with no structural homology to other proteins. The subunits of cyanase are arranged in a novel manner both at the dimer and decamer level. The dimer structure reveals the C-terminal domains to be intertwined, and the decamer is formed by a pentamer of these dimers. The active site of the enzyme is located between dimers and is comprised of residues from four adjacent subunits of the homodecamer. The structural data allow a conceivable reaction mechanism to be proposed. << Less

  Structure 8:505-514(2000) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Some properties of the induced enzyme cyanase.

  Taussig A.

  Can J Biochem 43:1063-1069(1965) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.