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C T Walsh

Publications and source records attributed to C T Walsh.

At least 271 records · Page 15Linked to original sources
PubMed1989

Nucleotide sequence of a cluster of Escherichia coli enterobactin biosynthesis genes: identification of entA and purification of its product 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase.

The nucleotide sequence of a region of the Escherichia coli chromosome encoding part of a cluster of genes involved in the biosynthesis of the iron chelator enterobactin has been determined. Four closely linked open reading frames, corresponding to the coding regions of entE (carboxy-terminal 144 amino acids), entB (32,554 daltons), entA (26,249 daltons), and an unidentified gene (P15) encoding a 14,970-dalton protein, were found. The lack of intergenic sequences and promoterlike elements suggests that these genes form part of the same transcription unit. We report the purification to homogeneity of the entA product, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase. It is an octamer of native molecular weight 210,000; the amino-terminal amino acid sequence confirmed the entA coding region. No isochorismate synthase activity was associated with this polypeptide. This finding leads to the conclusion that the recent suggestion (M. S. Nahlik, T. P. Fleming, and M. A. McIntosh, J. Bacteriol. 169:4163-4170, 1987) that 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase and isochorismate synthase activities reside on a single 26,000-dalton bifunctional enzyme is incorrect, even though the entA and entC mutations map to the same genetic locus.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of mutations in the HXK2 gene of Saccharomyces cerevisiae.

Several hundred new mutations in the gene (HXK2) encoding hexokinase II of Saccharomyces cerevisiae were isolated, and a subset of them was mapped, resulting in a fine-structure genetic map. Among the mutations that were sequenced, 35 were independent missense mutations. The mutations were obtained by mutagenesis of cloned HXK2 DNA carried on a low-copy-number plasmid vector and screened for a number of different phenotypes in yeast strains bearing chromosomal hxk1 and hxk2 null mutations. Some of these mutants were characterized both in vivo and in vitro; they displayed a wide spectrum of residual hexokinase activities, as indicated by three assays: in vitro enzyme activity, ability to grow on glucose and fructose, and ability to repress invertase production when growing on glucose. Of those that failed to support growth on fructose, only a small minority made normal-size, stable, and inactive protein. Analysis of the amino acid changes in these mutants in light of the crystallographically determined three-dimensional structure of hexokinase II suggests important roles in structure or catalysis for six amino acid residues, only two of which are near the active site.

Amino Acids↗
PubMed1989

The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.

Alleles↗
PubMed1989

Effects of Bay o 2752, a hypocholesterolemic agent, on intestinal taurocholate absorption and cholesterol esterification.

Bay o 2752 [N,N'-(1, 11-undecandiyl)bis(2,3-dihydro-2-methyl-1H-indole-1-carboxamide)] has been demonstrated in rats to inhibit intestinal cholesterol absorption. Studies were carried out in male Wistar rats to determine if the mechanism is inhibition of intestinal bile acid absorption or cholesterol esterification. Bay o 2752 did not alter intestinal bile acid absorption as measured by in vitro uptake of [14C]taurocholic acid into ileal everted sacs (0.01 and 1.0 mg/ml of Bay o 2752) or the biliary excretion of radioactivity after in vivo ileal perfusion of the bile acid and drug (1.0 mg/ml at 1.0 ml/min for 1 hr). Cholesterol esterification was determined by measurement of in vitro activity of acyl coenzyme A:cholesterol acyltransferase from hepatic microsomes and cholesterol ester hydrolase from pancreatic supernatant, and the in vivo lymphatic output of cholesteryl ester after intraduodenal cholesteryl infusion. Addition of Bay o 2752 (0.01-10 micrograms/ml) to hepatic microsomes produced a concentration-dependent decrease in acyl coenzyme A:cholesterol acyltransferase activity with an IC50 of 0.95 micrograms/ml. Cholesterol ester hydrolase activity was unaffected by the drug (1.0-100 micrograms/ml). Intraduodenal infusion of Bay o 2752 (10 mg/ml at 0.9 ml/hr for 8 hr) reduced markedly the flux of cholesterol from the intestinal lumen into the mesenteric lymph, especially the lymphatic output of the esterified form of both radioisotopically labeled and total cholesterol. These data suggest that Bay o 2752-induced reduction in intestinal cholesterol absorption results from its potent inhibitory effect on acyl coenzyme A:cholesterol acyltransferase activity.

Animals↗
PubMed1988

Kinetic characterization of the carbon monoxide-acetyl-CoA (carbonyl group) exchange activity of the acetyl-CoA synthesizing CO dehydrogenase from Clostridium thermoaceticum.

CO dehydrogenase from Clostridium thermoaceticum is a nickel-containing enzyme that catalyzes both the reversible conversion of CO2 to CO (for incorporation into the carbonyl group of acetate) and the synthesis of acetyl-CoA from methyl corrinoid, CO, and CoASH. The latter activity is conveniently assayed by monitoring the exchange of [1-14C]acetyl-CoA (carbonyl group) with 12CO. Kinetic parameters for the highly oxygen sensitive exchange activity have been determined: Km (acetyl-CoA) = 600 microM; Vmax = 440 min-1. In addition, coenzyme A analogues have been tested as inhibitors of the exchange to probe the active site of the enzyme; each has no effect on the CO2 in equilibrium CO activity of CO dehydrogenase. Coenzyme A, the substrate for acetate biosynthesis, is a potent competitive inhibitor, KI = 7 microM. Comparison of this value with that for desulfo-CoA (KI = 6000 microM) suggests that a key mode of binding is through the sulfur atom, possibly to a metal site on the enzyme. The relatively high affinity of the enzyme for CoASH relative to acetyl-CoA is consistent with its proposed operation in the acetogenic direction. The differential sensitivity to oxygen and storage of the two activities of CO dehydrogenase as well as the contrasting effect of coenzyme A inhibitors suggests that acetate assemblage occurs at a site distinct from that for CO dehydrogenation.

Acetate-CoA Ligase↗
PubMed1988

Inhibition of alanine racemase by alanine phosphonate: detection of an imine linkage to pyridoxal 5'-phosphate in the enzyme-inhibitor complex by solid-state 15N nuclear magnetic resonance.

Inhibition of alanine racemase from the Gram-positive bacterium Bacillus stearothermophilus by (1-aminoethyl) phosphonic acid (Ala-P) proceeds via a two-step reaction pathway in which reactivation occurs very slowly. In order to determine the mechanism of inhibition, we have recorded low-temperature, solid-state 15N NMR spectra from microcrystals of the [15N]Ala-P-enzyme complex, together with spectra of a series of model compounds that provide the requisite database for the interpretation of the 15N chemical shifts. Proton-decoupled spectra of the microcrystals exhibit a line at approximately 150 ppm, which conclusively demonstrates the presence of a protonated Ala-P-PLP aldimine and thus clarifies the structure of the enzyme-inhibitor complex. We also report the pH dependence of Ala-P binding to alanine racemase.

Alanine Racemase↗
PubMed1988

Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase.

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.

Amino Acid Sequence↗
PubMed1988

Cloning and characterization of a photolyase gene from the cyanobacterium Anacystis nidulans.

A 2 kb fragment was isolated from an Anacystis nidulans genomic DNA library by hybridization with synthetic oligonucleotide probes derived from the N-terminal amino acid sequence of Anacystis photolyase. This fragment contains a 1452 bp-long open reading frame encoding a polypeptide of 484 amino acids (Mr 54475). Antibodies raised against purified Anacystis photolyase reacted with extracts of cells harboring fused genes between lacZ of Escherichia coli and this gene. A 40.7% similarity was found between the deduced amino acid sequences of Anacystis and E. coli photolyases, notwithstanding the difference in chromophore structure.

Amino Acid Sequence↗
PubMed1988

Isolation, cloning, and sequencing of the Salmonella typhimurium ddlA gene with purification and characterization of its product, D-alanine:D-alanine ligase (ADP forming).

A gene coding for D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) (EC 6.3.2.4) activity has been isolated from a lambda library of Salmonella typhimurium DNA. Insertion mutations in the gene indicate that the gene is not essential for growth of the bacterium. The encoded enzyme was purified from an overproducing strain of S. typhimurium. D-Ala-D-Ala ligase is a protein of 39,271 molecular weight and has a kcat of 644 min-1 at pH 7.2. A 2.4-kilobase SalI-SphI fragment containing the gene was sequenced, and the ddlA gene consists of 1092 nucleotides. The gene sequence was compared to the sequence of the ddl gene of Escherichia coli [Robinson, A. C., Kenan, D. J., Sweeney, J., & Donachie, W. D. (1986) J. Bacteriol. 167, 809-817]. Because of differences between the S. typhimurium gene and the E. coli ddl gene, the S. typhimurium gene has been named ddlA.

Amino Acid Sequence↗
PubMed1988

ATP-dependent inactivation and slow binding inhibition of Salmonella typhimurium D-alanine:D-alanine ligase (ADP) by (aminoalkyl)phosphinate and aminophosphonate analogues of D-alanine.

In Salmonella typhimurium, D-alanine:D-alanine ligase (ADP) (EC 6.3.2.4) is the second enzyme in the three enzyme D-alanine branch pathway of peptidoglycan biosynthesis. The interaction of this enzyme with a possible transition-state analogue, the (aminoalkyl)phosphinate D-3-[(1-aminoethyl)phosphinyl]-2-heptylpropionic acid [Parsons et al. (1987) Abstracts of Papers, 193rd National Meeting of the American Chemical Society, Denver, CO, MEDI 63, American Chemical Society, Washington, DC], has been studied. This compound is a potent active site directed inhibitor and is competitive with D-alanine (Ki = 1.2 microM); it exhibits time-dependent inhibition in the presence of ATP. Kinetic analysis revealed a rapid onset of steady-state inhibition (kon = 1.35 X 10(4) M-1 s-1) followed by slow dissociation of inhibitory complex(es) with a half-life of 8.2 h. The inhibitory complex was shown to consist of E...I...ATP in equilibrium with E...I, Pi, and ADP. Similar time-dependent inhibition was also observed with D-(1-aminoethyl)phosphonic acid (D-Ala-P) (Ki = 0.5 mM; kon = 27 M-1 s-1; t1/2 for regain = 1.73 min) but not with D-(1-aminoethyl)phosphinic acid, which behaved as a simple competitive inhibitor (Ki = 0.4 mM). The mechanism of inhibition is discussed in the light of the precedents of glutamine synthase inhibition by methionine sulfoximine and phosphinothricin.

Alanine↗
PubMed1988

The NADPH-linked acetoacetyl-CoA reductase from Zoogloea ramigera. Characterization and mechanistic studies of the cloned enzyme over-produced in Escherichia coli.

The NADPH-linked acetoacetyl-CoA reductase, (R)-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36), from the bacterium Zoogloea ramigera, involved in the formation of D-3-hydroxybutyryl-CoA for poly(D-3-hydroxybutyrate) biosynthesis, has been purified from an over-producing Escherichia coli strain. The purification was achieved in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/mg). The enzyme is an alpha 4 homotetramer of four 25-kDa subunits. It has a Km of 2 microM and a kcat/Km of 1.8 X 10(8) M-1 s-1 for acetoacetyl-CoA; it is inhibited by acetoacetyl-CoA above 10 microM. K is 10(-10) M for the dehydrogenation. Kinetic studies of the back reaction revealed a sequential mechanism involving a ternary complex. The stereospecificity of the hydride-equivalent transfer was demonstrated using NMR techniques to be 4S (B side). Using the fingerprint method proposed by Wierenga et al. [(1986) J. Mol. Biol. 187, 101-107], we identified a 28-residue stretch (residues 3-31) as a possible NADPH fold. Finally the specificity of the reductase was examined using 3-oxo-acyl-CoA analogs and analogs lacking the adenosine 3',5'-bisphosphate moiety of CoA. Only the straight-chain C5 analog (3-oxo-propionyl-CoA) was found to be an alternative substrate (40%) for the reductase.

Alcohol Oxidoreductases↗
PubMed1988

Racemization of alanine by the alanine racemases from Salmonella typhimurium and Bacillus stearothermophilus: energetic reaction profiles.

Alanine racemases are bacterial pyridoxal 5'-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L----D and D----L directions for all three enzymes to assess the degree to which abstraction of the alpha-proton or protonation of substrate PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of alpha-3H from substrate to product and solvent exchange/substrate conversion experiments in 3H2O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis.

Alanine↗
PubMed1988

Reversible conversion of coenzyme F420 to the 8-OH-AMP and 8-OH-GMP esters, F390-A and F390-G, on oxygen exposure and reestablishment of anaerobiosis in Methanobacterium thermoautotrophicum.

Intracellular levels of F390 (AMP and GMP adducts of the 5-deazaflavin cofactor F420) in Methanobacterium thermoautotrophicum were analysed after gasing fermenter cultures with several consecutive cycles of substrate gas and gas mixtures containing 5% oxygen. No F390 was detected in growing cells, hydrogen starved cells and CO2 starved cells prior to O2 contamination. Also, no F390 was found in hydrogen depleted cells after O2 treatment. Exposure of exponentially growing cells and CO2 starved cells to oxygen lead to the formation of F390 species; the increase in the detected amount of F390 was coupled to a decrease of the F420 level. As soon as anaerobiosis was reestablished F390 cofactors were degraded and growth proceeded. Independent of the physiological condition of Methanobacterium thermoautotrophicum methanopterin was formed upon O2 exposure. After normal growth conditions were restored the level of detected methanopterin decreased again.

Anaerobiosis↗
PubMed1988

Molecular basis of bacterial resistance to organomercurial and inorganic mercuric salts.

Bacteria mediate resistance to organomercurial and inorganic mercuric salts by metabolic conversion to nontoxic elemental mercury, Hg(0). The genes responsible for mercury resistance are organized in the mer operon, and such operons are often found in plasmids that also bear drug resistance determinants. We have subcloned three of these mer genes, merR, merB, and merA, and have studied their protein products via protein overproduction and purification, and structural and functional characterization. MeR is a metalloregulatory DNA-binding protein that acts as a repressor of both its own and structural gene transcription in the absence of Hg(II); in addition it acts as a positive effector of structural gene transcription when Hg(II) is present. MerB, organomercury lyase, catalyzes the protonolytic fragmentation of organomercurials to the parent hydrocarbon and Hg(II) by an apparent SE2 mechanism. MerA, mercuric ion reductase, is an FAD-containing and redox-active disulfide-containing enzyme with homology to glutathione reductase. It has evolved the unique catalytic capacity to reduce Hg(II) to Hg(0) and thereby complete the detoxification scheme.

Bacteria↗
PubMed1988

Acinetobacter cyclohexanone monooxygenase: gene cloning and sequence determination.

The gene coding for cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871 was isolated by immunological screening methods. We located and determined the nucleotide sequence of the gene. The structural gene is 1,626 nucleotides long and codes for a polypeptide of 542 amino acids; 389 nucleotides 5' and 108 nucleotides 3' of the coding region are also reported. The complete amino acid sequence of the enzyme was derived by translation of the nucleotide sequence. From a comparison of the amino acid sequence with consensus sequences of nucleotide-binding folds, we identified a potential flavin-binding site at the NH2 terminus of the enzyme (residues 6 to 18) and a potential nicotinamide-binding site extending from residue 176 to residue 208 of the protein. An overproduction system for the gene to facilitate genetic manipulations was also constructed by using the tac promoter vector pKK223-3 in Escherichia coli.

Acinetobacter↗
PubMed1987

Specific proteolysis of native alanine racemases from Salmonella typhimurium: identification of the cleavage site and characterization of the clipped two-domain proteins.

Native DadB and Alr alanine racemases (Mr 39,000) from Salmonella typhimurium are proteolyzed at homologous positions by alpha-chymotrypsin, trypsin, and subtilisin to generate in all cases two nonoverlapping polypeptides of Mr 28,000 and 11,000. Under nondenaturing conditions, chymotryptic digest results in an associated form of the two fragments which possesses 3% of the original catalytic activity, incorporates 0.76 equiv of the mechanism-based inactivator beta-chloro-[14C]-D-alanine [Badet, B., Roise, D., & Walsh, C. T. (1984) Biochemistry 23, 5188], and exhibits a UV circular dichroism profile identical with that of native enzyme. Protein sequence analysis of the denatured chymotryptic fragments indicates the presence of a tetrapeptide interdomain hinge (DadB, residues 254-257; Alr, residues 256-259) that is attacked at both ends during proteolysis. Under the previously employed digest conditions, NaB3H4-reduced DadB holoenzyme is resistant to alpha-chymotrypsin and trypsin and is labile only toward subtilisin. These data suggest that the hinge structure is essential for a catalytically efficient enzyme species and is sensitive to active site geometry. The sequence at the hinge region is also conserved in alanine racemases from Gram-positive bacteria.

Alanine Racemase↗
PubMed1987

X-ray crystallographic studies of the alanine-specific racemase from Bacillus stearothermophilus. Overproduction, crystallization, and preliminary characterization.

To facilitate large-scale purification and crystallographic study, we have subcloned the gene for the alanine racemase of Bacillus stearothermophilus from pICR401 (Inagaki, K., Tanizawa, K., Badet, B., Walsh, C. T., Tanaka, H., and Soda, K. (1986) Biochemistry 25, 3268-3274) and overproduced the enzyme in Escherichia coli W3110 lacIq using the tac promoter of PKK223-3. This system yields alanine racemase as 6% of the bacterial cytosolic protein. Purification by a modification of the procedure of Inagake et al. yielded 75 mg of homogeneous alanine racemase from 30 g of cells (wet weight). Large, well-formed crystals of alanine racemase have been grown from polyethylene glycol 8000 using vapor diffusion. These crystals have unit cell dimensions a = 85.3 A, b = 110.0 A, and c = 89.9 A. The crystals belong to space group P2(1), with beta fortuitously equal to 90 degrees within experimental error; however, they are frequently twinned by second order pseudomerohedry with twin fraction (the ratio of the volume of the smaller twin domain to the total volume of the crystal) ranging from about 0 to 0.5. Fortunately, for crystals with low twin fraction, computational methods have been developed for the analysis and correction of simple twinning (Fisher, R. G., and Sweet, R. M. (1980) Acta Crystallogr. A36, 755-760). The crystals contain two alpha 2 dimers of alanine racemase in the asymmetric unit. We have identified several potentially useful heavy atom derivatives in low resolution screening experiments and are proceeding with high resolution data collection.

Alanine Racemase↗