A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli.

Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and delta cynT cynX::kan) did not differ from the parental strains with respect to cyanate sensitivity, presence of carbonic anhydrase and cyanase, or degradation of cyanate by whole cells; the physiological role of the cynX product remains unknown.     

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

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.     

Carbonic anhydrase in Escherichia coli. A product of the cyn operon.

The product of the cynT gene of the cyn operon in Escherichia coli has been identified as a carbonic anhydrase. The cyn operon also includes the gene cynS, encoding the enzyme cyanase. Cyanase catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The carbonic anhydrase was isolated from an Escherichia coli strain overexpressing the cynT gene and characterized. The purified enzyme was shown to contain 1 Zn2+/subunit (24 kDa) and was found to behave as an oligomer in solution; the presence of bicarbonate resulted in partial dissociation of the oligomeric enzyme. The kinetic properties of the enzyme are similar to those of carbonic anhydrases from other species, including inhibition by sulfonamides and cyanate. The amino acid sequence shows a high degree of identity with the sequences of two plant carbonic anhydrases. but not with animal and algal carbonic anhydrases. Since carbon dioxide formed in the bicarbonate-dependent decomposition of cyanate diffuses out of the cell faster than it would be hydrated to bicarbonate, the apparent function of the induced carbonic anhydrase is to catalyze hydration of carbon dioxide and thus prevent depletion of cellular bicarbonate.     

Identification and characterization of a cyanate permease in Escherichia coli K-12.

Escherichia coli contains an inducible enzyme, cyanase, that catalyzes the decomposition of cyanate into ammonia and bicarbonate. The gene encoding cyanase, cynS, was cloned and found to be on a DNA fragment that contained the lac operon. Characterization of a plasmid encoding cyanase indicated that a 26-kilodalton (kDa) protein of unknown function was also induced by cyanate (Y-C. Sung, D. Parsell, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 169:2639-2642, 1987). The gene encoding the 26-kDa protein was located between cynS and its promoter, indicating the existence of a cyn operon. The 26-kDa protein was identified as a cyanate permease that transports exogenous cyanate by active transport. E. coli was shown to contain a cyanate transport system that is energy dependent and saturable by cyanate.     

Reversible dissociation of active octamer of cyanase to inactive dimer promoted by alteration of the sulfhydryl group

Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate resulting in the decomposition of cyanate to ammonia and bicarbonate. In this study, the role of the single sulfhydryl group in each of the eight identical subunits of cyanase was investigated. Tetranitromethane, methyl methanethiosulfonate, N-ethylmaleimide, and Hg2+ all reacted with the sulfhydryl group to give derivatives which had reduced activities and which dissociated reversibly to inactive dimer. Association of inactive dimer to active octamer was facilitated by the presence of azide (cyanate analog) and bicarbonate, increased temperature and enzyme concentration, and presence of phosphate. Nitration of tyrosine residues by tetranitromethane occurred only in the absence of azide and bicarbonate, suggesting that at least some of the tyrosine residues become exposed when octamer dissociates to dimer. Site-directed mutagenesis was used to prepare a mutant enzyme in which serine was substituted for cysteine. The mutant enzyme was catalytically active and had properties very similar to native enzyme, except that it was less stable to treatment with urea and to high temperatures. These results establish that in native cyanase the sulfhydryl group per se is not required for catalytic activity, but it may play a role in stabilizing octameric structure, and that octameric structure is required for catalytic activity.     

Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase for the purpose of understanding the relationship between oligomeric structure and catalytic activity. Circular dichroism studies showed that cyanase has a significant amount of alpha-helix and beta-sheet structure. The one sulfhydryl group per subunit does not react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) unless cyanase is denatured. Denaturation is apparently complete in 10 M urea or 6 M guanidine hydrochloride, but is significantly reduced in 10 M urea by the presence of azide (analog of cyanate) and is incomplete in 8 M urea. Denatured cyanase could be renatured and reactivated (greater than 85%) by removal of denaturants. Reactivation was greatly facilitated by the presence of certain anions, particularly bicarbonate, and by high ionic strength and protein concentration. The catalytic activity of renatured cyanase was associated only with oligomer. Cyanase that had been denatured in the presence of DTNB to give a cyanase-DTNB derivative could also be renatured at 26 degrees C to give active cyanase-DTNB oligomer. The active oligomeric form of the cyanase-DTNB derivative could be converted reversibly to inactive dimer by lowering the temperature to 4 degrees C or by reduction of the ionic strength and removal of monoanions. These results provide evidence that free sulfhydryl groups are not required for catalytic activity and that catalytic activity may be dependent upon oligomeric structure.     

Proton transport by phosphate diffusion--a mechanism of facilitated CO2 transfer

We have measured CO2 fluxes across phosphate solutions at different carbonic anhydrase concentrations, bicarbonate concentration gradients, phosphate concentrations, and mobilities. Temperature was 22-25 degrees C, the pH of the phosphate solutions was 7.0-7.3. We found that under physiological conditions of pH and pCO2 a facilitated diffusion of CO2 occurs in addition to free diffusion when (a) sufficient carbonic anhydrase is present, and (b) a concentration gradient of HCO3- is established along with a pCO2 gradient, and (c) the phosphate buffer has a mobility comparable to that of bicarbonate. When the phosphate was immobilized by attaching 0.25-mm-long cellulose particles, no facilitation of CO2 diffusion was detectable. A mechanism of facilitated CO2 diffusion in phosphate solutions analogous to that in albumin solutions was proposed on the basis of these findings: bicarbonate diffusion together with a facilitated proton transport by phosphate diffusion. A mathematical model of this mechanism was formulated. The CO2 fluxed predicted by the model agree quantitatively with the experimentally determined fluxes. It is concluded that a highly effective proton transport mechanism acts in solutions of mobile phosphate buffers. By this mechanism; CO2 transfer may be increased up to fivefold and proton transfer may be increased to 10,000-fold.     

Indispensability of the Escherichia coli carbonic anhydrases YadF and CynT in cell proliferation at a low CO2 partial pressure

We found that a carbonic anhydrase, YadF, is essential for cell growth in the absence of another carbonic anhydrase, CynT, in Escherichia coli. However, mutant strains lacking both of them grew at high CO2 concentrations (5%), where non-enzymatic mechanisms generate HCO3-. This suggests that these carbonic anhydrases are essential because they maintain HCO3- levels at ambient CO2 concentrations.     

Kinetic properties of cyanase

Cyanase is an inducible enzyme in Escherichia coli that catalyzes the hydrolysis of cyanate. Bicarbonate is required for activity, perhaps as a substrate, and the initial product of the reaction is carbamate, which spontaneously breaks down to ammonia and bicarbonate [Anderson, P. M. (1980) Biochemistry 19, 2882]. The purpose of this study was to characterize the kinetic properties of cyanase. Initial velocity studies showed that both cyanate and bicarbonate act as competitive substrate inhibitors. A number of monovalent anions act as inhibitors. Azide and acetate appear to act as competitive inhibitors with respect to cyanate and bicarbonate, respectively. Chloride, bromide, nitrate, nitrite, and formate also inhibit, apparently as the result of binding at either substrate site. Malonate and several other dicarboxylic dianions at very low concentrations display "slow-binding", reversible inhibition which can be prevented by saturating concentrations of either substrate. The results are consistent with a rapid equilibrium random mechanism in which bicarbonate acts as a substrate, bicarbonate and cyanate bind at adjacent anion-binding sites, and both substrates can bind at the other substrate anion binding site to give a dead-end complex.     

Bicarbonate is a recycling substrate for cyanase

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.     

Involvement of the cynABDS operon and the CO2-concentrating mechanism in the light-dependent transport and metabolism of cyanate by cyanobacteria

The cyanobacteria Synechococcus elongatus strain PCC7942 and Synechococcus sp. strain UTEX625 decomposed exogenously supplied cyanate (NCO-) to CO2 and NH3 through the action of a cytosolic cyanase which required HCO3- as a second substrate. The ability to metabolize NCO- relied on three essential elements: proteins encoded by the cynABDS operon, the biophysical activity of the CO2-concentrating mechanism (CCM), and light. Inactivation of cynS, encoding cyanase, and cynA yielded mutants unable to decompose cyanate. Furthermore, loss of CynA, the periplasmic binding protein of a multicomponent ABC-type transporter, resulted in loss of active cyanate transport. Competition experiments revealed that native transport systems for CO2, HCO3-, NO3-, NO2-, Cl-, PO4(2-), and SO4(2-) did not contribute to the cellular flux of NCO- and that CynABD did not contribute to the flux of these nutrients, implicating CynABD as a novel primary active NCO- transporter. In the S. elongatus strain PCC7942 DeltachpX DeltachpY mutant that is defective in the full expression of the CCM, mass spectrometry revealed that the cellular rate of cyanate decomposition depended upon the size of the internal inorganic carbon (Ci) (HCO3- + CO2) pool. Unlike wild-type cells, the rate of NCO- decomposition by the DeltachpX DeltachpY mutant was severely depressed at low external Ci concentrations, indicating that the CCM was essential in providing HCO3- for cyanase under typical growth conditions. Light was required to activate and/or energize the active transport of both NCO- and Ci. Putative cynABDS operons were identified in the genomes of diverse Proteobacteria, suggesting that CynABDS-mediated cyanate metabolism is not restricted to cyanobacteria.     

Equilibrium of CO2 reactions in the pulmonary capillary

Steady-state CO2 excretion was measured in isolated blood-free rabbit lungs perfused with bicarbonate solutions. CO2 in the expired ventilation was either present initially in the perfusate as dissolved CO2 or produced from bicarbonate during pulmonary capillary transit. The two components were separated by measurement of simultaneous acetylene excretion. Bovine carbonic anhydrase and acetazolamide were sequentially added to the perfusate to determine the effects of maximal enzyme catalysis and inhibition of native lung carbonic anhydrase on CO2 production. Control CO2 production was significantly greater than that observed during inhibition of native lung carbonic anhydrase, confirming previous observations that bicarbonate has access to the tissue enzyme. Addition of excess carbonic anhydrase increased CO2 production by a statistically, but not physiologically, significant amount. These data demonstrate that CO2 reactions outside the erythrocyte attain 97% completion during pulmonary capillary transit. Under control and catalyzed conditions, alveolar and venous CO2 tens ions and pH were essentially identical to equilibrium values determined by in vitro tonometry.     

Sarcolemmal carbonic anhydrase in red and white rabbit skeletal muscle

Sarcolemmal vesicles of white and red skeletal muscles of the rabbit were prepared by consecutive density gradient centrifugations in sucrose and dextran according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13,862-13,871). White and red muscle membrane fractions enriched in sarcolemma were characterized by high ouabain-sensitive Na+, K(+)-ATPase, by high Mg2(+)-ATPase activity, and by a high cholesterol content. Ca2(+)-ATPase activity, a marker enzyme for sarcoplasmic reticulum, was not detectable in the highly purified white and red muscle sarcolemmal fractions. White and red muscle sarcolemmal fractions exhibited no significant differences with regard to Na+, K(+)-ATPase, Mg2(+)-ATPase, and cholesterol. Specific activity of carbonic anhydrase in white muscle sarcolemmal fractions was 38 U.ml/mg and was 17.6 U.ml/mg in red muscle sarcolemma. Inhibition properties of sarcolemmal carbonic anhydrase were analyzed for acetazolamide, chlorzolamide, and cyanate. White muscle sarcolemmal carbonic anhydrase is characterized by inhibition constants, KI, toward acetazolamide of 4.6 X 10(-8) M, toward chlorzolamide of 0.75 X 10(-8) M, and toward cyanate of 1.3 X 10(-4) M. Red muscle sarcolemmal carbonic anhydrase is characterized by KI values toward acetazolamide of 8.1 X 10(-8) M, toward chlorzolamide of 6.3 X 10(-8) M, and toward cyanate of 0.81 X 10(-4) M. In contrast to the high specific carbonic anhydrase activities in sarcolemma, carbonic anhydrase activity in sarcoplasmic reticulum from white muscle varied between values of only 0.7 and 3.3 U.ml/mg. Carbonic anhydrase of red muscle sarcoplasmic reticulum ranged from 2.4 to 3.7 U.ml/mg.     

Interaction of mono- and dianions with cyanase: evidence for apparent half-site binding

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent hydrolysis of cyanate. The dianions oxalate, oxalacetate, and malonate are slow-binding inhibitors of cyanase, and some monoanions such as azide and chloride also inhibit cyanase activity [Anderson, P. M., & Little, R. M. (1986) Biochemistry 25, 1621-1626]. The purpose of this study was to investigate the interaction of selected dianions and monoanions by kinetic and equilibrium dialysis binding studies in an effort to obtain information about the active site and catalytic mechanism. Measurement of the effectiveness of 30 different dianions as inhibitors of cyanase showed a significant degree of structural and/or isomeric specificity and considerable variation with respect to the slow-binding nature of the inhibition. Oxalate and oxalacetate both show extreme slow-binding inhibition at very low concentrations. Kinetic studies of the rate of inhibition of cyanase by oxalate showed that the reaction is pseudo first order with respect to oxalate concentration and the results are consistent with a pathway in which oxalate forms a complex with the enzyme in a rapid initial reversible step followed by a slow isomerization step leading to a complex with a very low dissociation constant. The rate of inhibition is significantly reduced by the presence of relatively low concentrations of either azide (analogue of cyanate) or bicarbonate. Equilibrium dialysis binding studies showed that the stoichiometry of binding at saturation for oxalate, malonate, chloride, and bicarbonate is about 0.5 mol of ligand bound/mol of subunit for each compound.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of Escherichia coli mutants lacking inducible cyanase

To determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated. Five independent mutations were localized next to lac; three of them lay between lacY and codA. Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt. Growth of the mutant stains was more sensitive to cyanate than growth of wild-type strains. This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (less than or equal to 1 mM). Higher concentrations inhibited growth of both wild-type and mutant strains. Urea in aqueous solutions dissociates slowly into ammonium cyanate. Accordingly wild-type strains were able to grow on a synthetic medium containing 0.5 M-urea whereas mutants lacking cyanase were not. We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.