Biomedical subjects

J A Fuchs

Publications and source records attributed to J A Fuchs.

At least 37 records · Page 2Linked to original sources

PubMed1994

Product binding modulates the thermodynamic properties of a Megasphaera elsdenii short-chain acyl-CoA dehydrogenase active-site mutant.

Previous work has shown that the redox properties of Megasphaera elsdenii short-chain acyl-CoA dehydrogenase (SCAD) are specifically modulated upon the binding of the substrate/product couple, allowing the reaction to proceed thermodynamically [Stankovich, M.T., & Soltysik, S. (1987) Biochemistry 26, 2627-2632]. The focus of this study on the Glu367Gln SCAD mutant protein is to gain an understanding of this phenomenon. The active-site mutant Glu367Gln SCAD inactivates the reductive and oxidative pathways and allows the effects of substrate (butyryl-CoA) and product (crotonyl-CoA) binding on the redox properties of the Glu367Gln SCAD mutant protein to be determined separately. Red anionic semiquinone was found to be thermodynamically stabilized in coulometric/potentiometric reductions of both butyryl-CoA- and crotonyl-CoA-complexed Glu367Gln SCAD. Reduction potential measurements showed that butyryl-CoA binding has little effect on the reduction potential of Glu367Gln SCAD. Crotonyl-CoA complexation, however, shifted the reduction potential of the Glu367Gln SCAD mutant protein by 30 mV in the positive direction. This modulation is similar to the 60-mV positive shift observed in native M. elsdenii SCAD upon complexation with the substrate/product couple [Stankovich, M.T., & Soltysik, S. (1987) Biochemistry 26, 2627-2632]. Thus, product binding and not substrate binding, thermodynamically regulates M. elsdenii SCAD. We propose that this observation is best explained by assuming that the product resembles an intermediate in the catalytic mechanism that is responsible for facilitating isopotential electron transfer from the substrate to the enzyme.

Acyl Coenzyme A↗

PubMed1994

Arsenate reductase of Staphylococcus aureus plasmid pI258.

Arsenate reductase encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 was overproduced in Escherichia coli and purified. The purified enzyme reduced radioactive arsenate to arsenite when coupled to thioredoxin, thioredoxin reductase, and NADPH. NADPH oxidation coupled to arsenate reduction also required thioredoxin and thioredoxin reductase. Glutaredoxin and reduced glutathione did not stimulate arsenate reduction. NADPH oxidation showed Michaelis-Menten kinetics with a Km of 1 microM AsO4(3-) and an apparent Vmax of 200 nmol/min per mg of protein. At high substrate concentration (above 1 mM AsO4(3-), a secondary rise in the reaction rate was observed, with a Km of 2 mM and an apparent Vmax of 450 nmol/min per mg of protein. This secondary rise also occurred upon addition of phosphate or nitrate (which were not substrates for the enzyme). Arsenite (the product of the enzyme), tellurite, and antimonite [Sb(III)] were inhibitors. Selenate (but not selenite or sulfate) was a substrate for reductase-dependent NADPH oxidation, with an apparent Km of 13 mM SeO4(2-). Arsenate reductase was purified as a monomer of 14.5 kDa, consistent with the DNA sequence. Electrospray mass spectrometry showed two molecular masses of 14,810.5 and 14,436.0 Da, suggesting that 70% of the purified protein lacked the N-terminal three amino acids; HPLC coupled to electrospray mass spectroscopy of protease digest products confirmed this conclusion and verified the entire amino acid sequence.

Adenosine Triphosphatases↗

PubMed1994

13C NMR and fluorescence analysis of tryptophan dynamics in wild-type and two single-Trp variants of Escherichia coli thioredoxin.

The rotational motion of tryptophan side chains in oxidized and reduced wild-type (WT) Escherichia coli thioredoxin and in two single-tryptophan variants of E. coli thioredoxin was studied in solution in the temperature range 20-50 degrees C from 13C-NMR relaxation rate measurements at 75.4 and 125.7 MHz and at 20 degrees C from steady-state and time-resolved trp fluorescence anisotropy measurements. Tryptophan enriched with 13C at the delta 1 and epsilon 3 sites of the indole ring was incorporated into WT thioredoxin and into two single-trp mutants, W31F and W28F, in which trp-28 or trp-31 of WT thioredoxin was replaced, respectively, with phenylalanine. The NMR relaxation data were interpreted using the Lipari and Szabo "model-free" approach (G. Lipari and A. Szabo. 1982. J. Amer. Chem. Soc. 104:4546-4559) with trp steady-state anisotropy data included for the variants at 20 degrees C. Values for the correlation time for the overall rotational motion (tau m) from NMR of oxidized and reduced WT thioredoxin at 35 degrees C agree well with those given by Stone et al. (Stone, M. J., K. Chandrasekhar, A. Holmgren, P. E. Wright, and H. J. Dyson. 1993. Biochemistry. 32:426-435) from 15N NMR relaxation rates, and the dependence of tau m on viscosity and temperature was in accord with the Stokes-Einstein relationship. Order parameters (S2) near 1 were obtained for the trp side chains in the WT proteins even at 50 degrees C. A slight increase in the amplitude of motion (decrease in S2) of trp-31, which is near the protein surface, but not of trp-28, which is partially buried in the protein matrix, was observed in reduced relative to oxidized WT thioredoxin. For trp-28 in W31F, order parameters near 1 (S2 > or = 0.8) at 20 degrees C were found, whereas trp-31 in W28F yielded the smallest order parameters (S2 approximately 0.6) of any of the cases. Analysis of time-resolved anisotropy decays in W28F and W31F yielded S2 values in good agreement with NMR, but gave tau m values about 60% smaller. Generally, values of tau e, the effective correlation time for the internal motion, were < or = 60 ps from NMR, whereas somewhat longer times were obtained from fluorescence. The ability of NMR and fluorescence techniques to detect subnanosecond motions in proteins reliably is examined.

Carbon Isotopes↗

PubMed1994

Regulation of the Escherichia coli nrd operon: role of DNA supercoiling.

An in vitro RNA transcription assay was used to investigate the regulation of the expression of the nrd promoter. Using a linear DNA template, we found that Fis protein, which has a positive effect on expression of the nrd promoter in an nrd-lacZ fusion in vivo, had a moderate negative effect in vitro. However, with a supercoiled DNA template as substrate, we found that Fis had a concentration-dependent positive effect on nrd transcription in vitro. This positive effect was not present on two templates that had 35- or 37-bp insertions between the Fis binding site and the nrd promoter. In the absence of Fis protein, a dramatic decrease in transcription was observed in templates with reduced supercoiling generated by the treatment with wheat germ topoisomerase I. Templates with insertions of 35 bp into an HpaII site at -102 or 37 bp into the MnlI site at -33 bp from the start of transcription failed to exhibit the DNA supercoiling sensitivity of the nrd promoter. Analysis of cells containing either of these two nrd-lacZ fusion constructs that has an insertion at the regulatory region by flow cytometry indicated that these two constructs, unlike the parental construct, were not cell cycle regulated.

Base Sequence↗

PubMed1994

Expression of proteins encoded by the Escherichia coli cyn operon: carbon dioxide-enhanced degradation of carbonic anhydrase.

Cyanase catalyzes the reaction of cyanate with bicarbonate to give 2CO2. The cynS gene encoding cyanase, together with the cynT gene for carbonic anhydrase, is part of the cyn operon, the expression of which is induced in Escherichia coli by cyanate. The physiological role of carbonic anhydrase is to prevent depletion of cellular bicarbonate during cyanate decomposition due to loss of CO2 (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). A delta cynT mutant strain was extremely sensitive to inhibition of growth by cyanate and did not catalyze decomposition of cyanate (even though an active cyanase was expressed) when grown at a low pCO2 (in air) but had a Cyn+ phenotype at a high pCO2. Here the expression of these two enzymes in this unusual system for cyanate degradation was characterized in more detail. Both enzymes were found to be located in the cytosol and to be present at approximately equal levels in the presence of cyanate. A delta cynT mutant strain could be complemented with high levels of expressed human carbonic anhydrase II; however, the mutant defect was not completely abolished, perhaps because the E. coli carbonic anhydrase is significantly less susceptible to inhibition by cyanate than mammalian carbonic anhydrases. The induced E. coli carbonic anhydrase appears to be particularly adapted to its function in cyanate degradation. Active cyanase remained in cells grown in the presence of either low or high pCO2 after the inducer cyanate was depleted; in contrast, carbonic anhydrase protein was degraded very rapidly (minutes) at a high pCO2 but much more slowly (hours) at a low pCO2. A physiological significance of these observations is suggested by the observation that expression of carbonic anhydrase at a high pCO2 decreased the growth rate.

Carbon Dioxide↗

PubMed1994

Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion.

The Escherichia coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase. The regulation of the nrd operon has been observed to be very complex. The specific binding of two proteins to the nrd regulatory region and expression of mutant nrd-lac fusions that do not bind these proteins are described. A partially purified protein from an E. coli cell extract was previously shown to bind to the promoter region and to regulate transcription of the nrd operon (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990). We have purified this protein to homogeneity by affinity chromatography and identified it as the E. coli factor for inversion stimulation (Fis). Cu-phenanthroline footprinting experiments showed that Fis binds to a site centered 156 bp upstream of the start of nrd transcription. Mutants with deletion and site-directed mutations that do not bind Fis at this site have two- to threefold-lower expression of an nrd-lac fusion. The previously reported negative regulatory nature of this site (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990) was found to be due to a change in polarity in the vectors used to construct promoter fusions. Two nine-base sequences with homology to the DnaA consensus binding sequence are located immediately upstream of the nrd putative -35 RNA polymerase binding site. Binding of DnaA to these sequences on DNA fragments containing the nrd promoter region was confirmed by in vitro Cu-phenanthroline footprinting. Footprinting experiments on fragments with each as well as both of the mutated 9-mers suggests cooperativity between the two sites in binding DnaA. Assay of in vivo expression from wild-type and DnaA box-mutated nrd promoter fragments fused to lacZ on single-copy plasmids indicates a positive effect of DnaA binding on expression of nrd.

Bacterial Proteins↗

PubMed1994

Functional analysis of the Escherichia coli K-12 cyn operon transcriptional regulation.

The cynTSX operon enables Escherichia coli K-12 to degrade and use cyanate as a sole nitrogen source. The promoter of this operon is positively regulated by cyanate and the CynR protein. CynR, a member of the LysR family of regulatory proteins, binds specifically to a 136-bp DNA fragment containing both the cynR and the cynTSX promoters. In this study, we report the results of DNase I digestion studies showing that CynR protects a 60-bp region on the cynR coding strand and a 56-bp sequence on the cynTSX coding strand. CynR binding was not affected by cyanate or its structural homolog azide, a gratuitous inducer of the operon. However, CynR-induced bending of two different DNA fragments was detected. The amount of bending was decreased by cyanate.

Azides↗

PubMed1994

Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence.

The expression of the nrd operon encoding ribonucleotide reductase in Escherichia coli has been shown to be cell cycle regulated. To identify the cis-acting elements required for the cell cycle regulation of the nrd promoter, different 5' deletions as well as site-directed mutations were translationally fused to a lacZ reporter gene. The expression of beta-galactosidase from these nrd-lacZ fusions in single-copy plasmids was determined with synchronously growing cultures obtained by repeated phosphate starvation as well as with exponentially growing cultures by flow cytometry analysis. Although Fis and DnaA, two regulatory proteins that bind at multiple sites on the E. coli chromosome, have been found to regulate the nrd promoter, the results in this study demonstrated that neither Fis nor DnaA was required for nrd cell cycle regulation. A cis-acting upstream AT-rich sequence was found to be required for the cell cycle regulation. This sequence could be replaced by a different sequence that maintained the AT richness. A flow cytometry analysis that combined specific immunofluorescent staining of beta-galactosidase with a DNA-specific stain was developed and employed to study the nrd promoter activity in cells at specific cell cycle positions. The results of the flow cytometry analysis confirmed the results obtained from studies with synchronized cells.

Base Sequence↗

PubMed1993

Characterization of wild-type and an active-site mutant in Escherichia coli of short-chain acyl-CoA dehydrogenase from Megasphaera elsdenii.

The objective of this work is to determine the molecular mechanism and regulation of short-chain acyl-CoA dehydrogenase (SCAD) from Megasphaera elsdenii. To achieve this, the gene coding for SCAD from M. elsdenii was cloned and sequenced. Site-directed mutagenesis was then used to identify an amino acid residue that is required for the proposed mechanism. To clone the gene, the amino acid sequence of the 50 N-terminal residues of SCAD from M. elsdenii was determined. This sequence information was utilized to synthesize two sets of mixed oligonucleotide primers which were then used to generate a 120-bp specific probe from M. elsdenii DNA by the polymerase chain reaction (PCR) method. The 120-bp probe was used to screen a M. elsdenii genomic DNA library cloned into Escherichia coli. The gene encoding M. elsdenii SCAD was identified from this library, sequenced, and expressed. The cloned SCAD gene contained an open reading frame which revealed a high degree of sequence identity with an open reading frame protein sequence of the human SCAD and the rat medium-chain acyl-CoA dehydrogenase (MCAD) (44% and 36% identical residues in paired comparisons for human SCAD and rat MCAD, respectively). Recombinant SCAD expressed from a pUC119 vector accounted for 35% of the cytosolic protein in the Escherichia coli crude extract. The expressed protein had similar activity, redox potential properties, and nearly identical amino acid composition to native M. elsdenii SCAD. In addition, a site-directed Glu367 Gln mutant of SCAD expressed from a pUC119 vector was shown to have minimal reductive and oxidative pathway activity with butyryl-CoA and crotonyl-CoA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl-CoA Dehydrogenase↗

PubMed1993

Hydrogen exchange identifies native-state motional domains important in protein folding.

Effects of mutations on hydrogen exchange kinetics, structure, and stability suggest that the slow exchange core is a key element in protein folding. Single amino acid variants of bovine pancreatic trypsin inhibitor (BPTI) have been made with glycine or alanine replacement of residues Tyr 35, Gly 37, Asn 43, and Asn 44. The crystal structures of Y35G and N43G are reported [Housset, D., Kim, K.-S., Fuchs, J., & Woodward, C. (1991) J. Mol. Biol. 220, 757-770; Danishefsky, A. T., Housset, D., Kim, K.-S., Tao, F., Fuchs, J., Woodward, C., & Wlodawer, A. (1993) Protein Sci. 2, 577-587; Kim, K.-S., Tao, F., Fuchs, J. A., Danishefsky, A. T., Housset, D., Wlodawer, A., & Woodward, C. (1993a) Protein Sci. 2, 588-596]. NMR chemical shifts indicate few changes from the wild type (WT) in G37A and N44G. Stabilities of the four mutants were measured by calorimetry and by hydrogen exchange. Values of delta delta(WT-->mut), the difference in delta G of folding/unfolding between the wild type and mutant, estimated by both methods are in good agreement and are in the range 4.7-6.0 kcal/mol. There is no general correlation between stability and hydrogen exchange rates at pH 3.5 and 30 degrees C. Exchange occurs by two parallel pathways, one involving small noncooperative fluctuations of the native state, and the other involving cooperative, global unfolding. In the mutant proteins, the rates for exchange by the unfolding mechanism are accelerated by a factor corresponding to the increase in the unfolding/folding equilibrium constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

The reported cDNA sequence for phospholipase C alpha encodes protein disulfide isomerase, isozyme Q-2 and not phospholipase-C.

We have previously shown that the cDNA sequence published for the rat gene encoding phosphatidylinositol specific phospholipase C alpha (1) may be identical to the sequence of a new isoform of protein disulfide isomerase, Isozyme Q2 (2). To determine whether the presumed phospholipase C alpha gene encodes phospholipase C or protein disulfide isomerase, we have now expressed the cDNA in Escherichia coli and find that the 55.5 kDa protein that it synthesized contains no phospholipase C activity. Moreover, an extract of Escherichia coli containing the expressed protein shows a concentration dependent increase in protein disulfide isomerase activity. The expressed protein is identical to protein disulfide isomerase, Isozyme Q2 on SDS-PAGE and by Western blot analysis. These current studies confirm that the published sequence of phospholipase C alpha is, in fact, the sequence of protein disulfide isomerase, Isozyme Q2.

Animals↗

PubMed1993

Determination of the pKa values of active-center cysteines, cysteines-32 and -35, in Escherichia coli thioredoxin by Raman spectroscopy.

We have determined the pKa values for thiol-thiolate equilibria of Escherichia coli thioredoxin and compared structural properties of reduced and oxidized forms of the protein in solution by Raman spectroscopy. Ionization and hydrogen-bonding states of the two cysteine sulfhydryls (Cys32 and Cys35) in reduced thioredoxin were determined by monitoring the complex Raman SH stretching band (Li & Thomas, 1991) as a function of pH in the range 4.0 < pH < 12.2. The Raman SH markers indicate the following: (i) Both sulfhydryls of the native protein are relatively robust hydrogen-bond donors, but one is a stronger hydrogen-bond donor than the other. (ii) The sulfhydryl which donates the weaker hydrogen bond, assigned tentatively to Cys32, is preferentially titrated to the thiolate ion (S-) as the solution pH is increased from 4 to 7. Water or a hydroxyl oxygen is the probable acceptor for the Cys32 S-H donor. (iii) The sulfhydryl which donates the stronger hydrogen bond, Cys35, resists substantial ionization until roughly 50% of the more acidic sulfhydryl has been titrated. A carbonyl oxygen is proposed as the likely acceptor of the Cys35 S-H donor. (iv) The Raman titration data indicate pK1 = 7.1 +/- 0.2 and pK2 = 7.9 +/- 0.2 for the two thiol-thiolate equilibria. The lower pKa, which is the more strongly perturbed, is assigned tentatively to Cys32.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Crystal structure analysis of a mutant Escherichia coli thioredoxin in which lysine 36 is replaced by glutamic acid.

The structure of a mutant Escherichia coli thioredoxin with a glutamic acid substituted for a conserved lysine at position 36 adjacent to the active site has been solved using molecular replacement and refined at 2.0-A resolution to a crystallographic residual of 19.9%. The mutant was crystallized in an orthorhombic space group with one molecule in the asymmetric unit. The structure of the mutant thioredoxin shows overall good agreement with the wild-type E. coli thioredoxin. The root-mean-square deviations for all C alpha s are 0.45 and 0.79 A between the mutant structure and the two molecules in the asymmetric unit of the wild-type crystals. Structural changes are seen in several residues in the active-site region preceding the disulfide. A reverse turn of residues 29-32 changes the conformation from a type I to a type II turn. This change may be related to the loss of a hydrogen bond from Lys-36 to the main-chain carbonyl of residue 30 due to the mutation. The C alpha atom of Trp-31 has moved 1.9 A and the indole ring no longer makes hydrogen bonds to the carboxyl group of Asp-61 but instead participates in a crystal contact. The structural differences seen in the mutant thioredoxin may be influenced by the crystal packing. The substituted Glu-36 makes extensive crystal contacts. The static fluorescence of this mutant thioredoxin has a different pH dependence than the wild type.

Binding Sites↗

PubMed1993

Expression and purification of the cynR regulatory gene product: CynR is a DNA-binding protein.

The CynR protein, a member of the LysR family, positively regulates the Escherichia coli cyn operon and negatively autoregulates its own transcription. By S1 mapping analysis, the in vivo cynR transcription start site was located 63 bp upstream of the cynTSX operon transcription start site. Topologically, the cynR and cynTSX promoters overlap and direct transcription in opposite directions. The CynR translation initiation codon was identified by oligonucleotide-directed mutagenesis, and the CynR coding sequence was cloned under the control of a T7 phage promoter. The CynR protein was stably expressed at a high level with a T7 RNA polymerase-T7 phage promoter system. Purification by ion-exchange chromatography, affinity chromatography, and ammonium sulfate fractionation yielded pure CynR protein. Gel shift assays confirmed that CynR is a DNA-binding protein like the other members of the LysR family. The CynR regulatory protein binds specifically to a 136-bp DNA fragment encompassing both the cynR and the cynTSX promoters.

Amino Acid Sequence↗

PubMed1993

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.

Bicarbonates↗

PubMed1993

Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypeptide. One copy of this gene was cloned, and its complete nucleotide sequence is presented. Immediately downstream of this gene, in the same operon, is the gene for a 40-kDa polypeptide that copurifies with the ammonia monooxygenase acetylene-binding polypeptide. The sequence of the first 692 nucleotides of this structural gene, coding for about two-thirds of the protein, is presented. These sequences are the first sequences of protein-encoding genes from an ammonia-oxidizing autotrophic nitrifying bacterium. The two protein sequences are not homologous with the sequences of any other monooxygenase. From radioactive labelling of ammonia monooxygenase with [14C]acetylene it was determined that there are 23 nmol of ammonia monooxygenase per g of cells. The kcat of ammonia monooxygenase for NH3 in vivo was calculated to be 20 s-1.

Acetylene↗

PubMed1993

Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea.

The genome of Nitrosomonas europaea contains at least three copies each of the genes coding for hydroxylamine oxidoreductase (HAO) and cytochrome c554. A copy of an HAO gene is always located within 2.7 kb of a copy of a cytochrome c554 gene. Cytochrome P-460, a protein that shares very unusual spectral features with HAO, was found to be encoded by a gene separate from the HAO genes.

Amino Acid Sequence↗

PubMed1992

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.

Carbon-Nitrogen Lyases↗