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T Toraya

Publications and source records attributed to T Toraya.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cloning, sequencing, and heterologous expression of rat methionine synthase cDNA.

Methionine synthase catalyzes cobalamin-dependent methyl transfer reaction from 5-methyltetrahydrofolate to homocysteine, forming methionine. Rat methonine synthase cDNA was cloned and analyzed by RT-PCR, 3'- and 5'-RACE techniques. The cDNA consists of a 0.3-kb upstream untranslated region, a 3.8-kb coding region, and a 0.4-kb downstream untranslated region. The open reading frame encoded a polypeptide of 1,253 amino acid residues with a calculated molecular weight of 139,162. This molecular weight was in good agreement with the observed one (143,000) of the purified rat liver enzyme. The deduced amino acid sequence was 53, 92, and 64% identical with those of the Escherichia coli, human, and presumptive Caenorhabditis elegans enzymes, respectively. All the fingerprint sequences, forming parts of the cobalamin- and S-adenosylmethionine-binding sites, were completely conserved in the rat methionine synthase. A high-level expression of catalytically active enzyme in insect cells was done by infection with a baculovirus containing the rat methionine synthase cDNA.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1997

Characterization, sequencing, and expression of the genes encoding a reactivating factor for glycerol-inactivated adenosylcobalamin-dependent diol dehydratase.

Diol dehydratase undergoes suicide inactivation by glycerol during catalysis involving irreversible cleavage of the Co-C bond of adenosylcobalamin. In permeabilized Klebsiella oxytoca and Klebsiella pneumoniae cells, the glycerol-inactivated holoenzyme or the enzyme-cyanocobalamin complex is rapidly activated by the exchange of the inactivated coenzyme or cyanocobalamin for free adenosylcobalamin in the presence of ATP and Mg2+ (Honda, S., Toraya, T., and Fukui, S. (1980) J. Bacteriol. 143, 1458-1465; Ushio, K., Honda, S., Toraya, T., and Fukui, S. (1982) J. Nutr. Sci. Vitaminol. 28, 225-236). Permeabilized Escherichia coli cells co-expressing the diol dehydratase genes with two open reading frames in the 3'-flanking region were capable of reactivating glycerol-inactivated diol dehydratase as well as activating the enzyme-cyanocobalamin complex in situ in the presence of free adenosylcobalamin, ATP, and Mg2+. These open reading frames, designated as ddrA and ddrB genes, were identified as the genes of a putative reactivating factor for inactivated diol dehydratase. The genes encoded polypeptides consisting of 610 and 125 amino acid residues with predicted molecular weights of 64,266 and 13,620, respectively. Co-expression of the open reading frame in the 5'-flanking region was stimulatory but not obligatory for conferring the reactivating activity upon E. coli. Thus, the product of this gene was considered not an essential component of the reactivating factor.

Amino Acid Sequence↗
PubMed1997

Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca.

Recombinant adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca overexpressed in Escherichia coli was purified to homogeneity. The enzyme has a low solubility and was extracted from the crude membrane fraction with 1% Brij 35 in a high recovery. Subsequent chromatography on DEAE-cellulose resulted in 4.9-fold purification of the enzyme in an overall yield of 65%. The enzyme thus obtained showed specific activity comparable to that of the wild-type enzyme of K. oxytoca. The apparent molecular weight determined by nondenaturing gel electrophoresis on a gradient gel was 220,000. The enzyme consists of equimolar amounts of the three subunits with apparent Mr of 60,000 (alpha), 30,000 (beta), and 19,000 (gamma). Therefore, the subunit structure of the enzyme is most likely alpha2beta2gamma2. The recombinant enzyme was also separated into components F and S upon DEAE-cellulose chromatography in the absence of substrate. Components F and S were identified as the beta subunit and alpha2gamma2 complex, respectively. Apparent Km for adenosylcobalamin, 1,2-propanediol, glycerol, and 1,2-ethanediol were 0.83 microM, 0.08 mM, 0.73 mM, and 0.56 mM, respectively. The three genes encoding the subunits of diol dehydratase were overexpressed individually or in various combinations in Escherichia coli. The alpha and gamma subunits mutually required each other for correct folding forming the soluble, active alpha2gamma2 complex (component S). Expression of the beta subunit in a soluble, active form (component F) was promoted by coexpression with both the alpha and gamma subunits, probably by coexistence with component S. These lines of evidence indicate that each subunit mutually affects the folding of the others in this heterooligomer enzyme.

Chromatography, Ion Exchange↗
PubMed1997

An electron paramagnetic resonance study on the mechanism-based inactivation of adenosylcobalamin-dependent diol dehydrase by glycerol and other substrates.

Adenosylcobalamin-dependent diol dehydrase undergoes mechanism-based inactivation by glycerol or other substrates during catalysis. X-band electron paramagnetic resonance spectra of holoenzyme were measured at -130 degrees C after reaction with such substrates. After short time of incubation, broad signals assigned to low-spin Co(II) of cob(II)alamin and doublet signals assigned to an organic radical intermediate derived from each substrate were observed with 1,2-propanediol, 1,2-ethanediol, glycerol and meso-2,3-butanediol with the magnitude of their exchange interaction (J-value) decreasing in this order. A substrate with the smaller magnitude of exchange interaction between low-spin Co(II) and an organic radical intermediate seems to be an efficient mechanism-based inactivator. Since the magnitude of exchange interaction decreases with the distance between radical species in a radical pair, these results suggest that a stabilizing effect of holoenzyme on radical intermediates during reactions decreases with the distance between Co(II) and a radical.

Apoenzymes↗
PubMed1997

A protein factor is essential for in situ reactivation of glycerol-inactivated adenosylcobalamin-dependent diol dehydratase.

The adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicidal inactivation by glycerol during catalysis involving irreversible dissociation of the Co-C bond of the coenzyme. The glycerol-inactivated holoenzyme in permeabilized cells (in situ) of E. coli harboring a plasmid containing the diol dehydratase genes and their flanking regions was rapidly reactivated in the presence of free AdoCbl, ATP, and Mg2+. beta,gamma-Methylene ATP was not able to replace ATP. Inactive complexes of the enzyme with aqCbl, CN-Cbl, and PeCbl were activated in situ in the presence of AdoCbl, ATP, and Mg2+, but the complex with AdePeCbl was not. These results suggest that the inactivated holoenzyme is reactivated in situ in the presence of ATP and Mg2+ by exchange of the inactivated coenzyme lacking the adenine moiety for free intact AdoCbl. The in situ reactivation was also observed when an analog lacking the alpha-ribose moiety of the nucleotide loop was used as coenzyme. The results with a recombinant E. coli strains carrying a deletion mutant plasmid demonstrate that certain protein(s) encoded by the 3'-flanking region of the diol dehydratase genes are essential for the in situ reactivation of inactivated diol dehydratase.

Adenosine Triphosphate↗
PubMed1997

Purification and some properties of cobalamin-dependent methionine synthase from rat liver.

Cobalamin-dependent methionine synthase was purified from rat liver. The enzyme activity was separated into two peaks upon Mono-Q column chromatography. Peaks I and II of the enzyme, eluted in this order, were purified 18,000- and 44,000-fold in overall yields of 0.7 and 1.8%, respectively. Peak II methionine synthase, the major fraction, was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. The enzyme was a large monomeric protein with an apparent molecular weight of 143,000 Da. Interconversion of the enzyme between the two peaks was not observed during purification procedures. The enzyme required S-adenosylmethionine and a reducing system for activity. Apparent K(m) values of the peak II enzyme for 5-methyltetrahydrofolate and homocysteine were 75 and 1.7 microM, respectively.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1996

Cloning, sequencing, and high level expression of the genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae.

The gld genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae were cloned by cross-hybridization with a DNA fragment of Klebsiella oxytoca diol dehydrase genes. Since the Escherichia coli clones isolated did not show appreciable enzyme activity, plasmids for high level expression of cloned genes were constructed. The enzyme expressed in E. coli was indistinguishable from the wild-type glycerol dehydrase of K. pneumoniae by the criteria of polyacrylamide gel electrophoretic, immunochemical, and catalytic properties. It was also shown that the recombinant functional enzyme consists of Mr 61,000, 22,000, and 16, 000 subunits. Sequence analysis of the genes revealed four open reading frames separated by 2-12 bases. The sequential three open reading frames from the first to the third (gldA, gldB, and gldC genes) encoded polypeptides of 555, 194, and 141 amino acid residues with predicted molecular weights of 60,659(alpha), 21,355(beta), and 16,104(gamma), respectively. High level expression of these three genes in E. coli produced more than 14-fold higher level of fully active apoenzyme than that in K. pneumoniae. It was thus concluded that these are the genes encoding the subunits of glycerol dehydrase. The deduced amino acid sequences of the three subunits were 71, 58, and 54% identical with those of the alpha, beta, and gamma subunits of diol dehydrase, respectively, but failed to show any apparent homology with other proteins.

Amino Acid Sequence↗
PubMed1995

Molecular cloning, sequencing, and expression of the genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca.

The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucleotide as a hybridization probe followed by measuring the enzyme activity of each clone. Five clones of Escherichia coli exhibited diol dehydrase activity. At least one of them was shown to express diol dehydrase genes under control of their own promoter. Sequence analysis of the DNA fragments found in common in the inserts of these five clones and the flanking regions revealed four open reading frames separated by 10-18 base pairs. The sequential three open reading frames from the second to the fourth (pddA, pddB, and pddC genes) encoded polypeptides of 554, 224, and 173 amino acid residues with predicted molecular weights of 60,348 (alpha), 24,113 (beta), and 19,173 (gamma), respectively. Overexpression of these three genes in E. coli produced more than 50-fold higher level of functional apodiol dehydrase than that in K. oxytoca. The recombinant enzyme was indistinguishable from the wild-type one of K. oxytoca by the criteria of polyacrylamide gel electrophoretic and immunochemical properties. It was thus concluded that these three gene products are the subunits of functional diol dehydrase. Comparisons of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.

Amino Acid Sequence↗
PubMed1995

Purification and structural determination of an inhibitor of starfish oocyte maturation from a Bacillus species.

Inhibitors of bacterial origins of starfish oocyte maturation were sought to obtain biologically active substances which act on either hormonal signal transduction or cell cycle regulation. An oocyte maturation-inhibiting substance found in culture fluid of a Bacillus species was purified to homogeneity. This substance possessed the nature of a detergent and specifically inhibited 1-methyladenine-induced oocyte maturation (50% inhibitory concentration, 3.3 microM) but not dithiothreitol-induced maturation. Its total structure was established to be the lactone of 3-hydroxy-13-methyltetradecanoyl-Glu-Leu-Leu-Val-Asp-Leu -Leu through COOH of the carboxy-terminal Leu. This structure is identical to surfactin, although although the configuration of the substance's amino acid residues has not yet been determined. Surfactin was shown to be identical with this substance in its inhibitory effect on starfish oocyte maturation as well as its chromatographic and electrophoretic properties. Therefore, it was concluded that the oocyte maturation-inhibiting substance produced by a Bacillus species is surfactin.

Adenine↗
PubMed1995

Synthesis, properties and microbiological activity of hydrophobic derivatives of vitamin B12.

Long chain alkylcobalamins and long chain acyl-cyanocobalamins, two types of hydrophobic derivatives of vitamin B12, were synthesized. It was shown by TLC and determination of the partition coefficient between organic and aqueous phases that the hydrophobicity of alkylcobalamins and acyl-cyanocobalamins increased with the chain length of the alkyl or acyl group introduced into cobalamin. Long chain alkylcobalamins were easily converted to aquacobalamin by photoirradiation, but the first-order rate constant of photolysis decreased with the length of an alkyl group. Long chain acyl-cyanocobalamins were gradually hydrolyzed to cyanocobalamin in neutral or alkaline solution with the pseudo-first order rate constant increasing with the pH of the solution. Stabilization of acyl-cyanocobalamins toward hydrolysis was achieved by introducing a methyl group into the alpha-position of an acyl group. All the long chain alkylcobalamins tested supported the growth of Escherichia coli 215, a cobalamin- or L-methionine-auxotroph, and Lactobacillus leichmannii, although their activity as cobalamin was at most 28% and 15% that of cyanocobalamin for E. coli 215 and L. leichmannii, respectively.

Acylation↗
PubMed1994

The synthesis of a pyridyl analog of adenosylcobalamin and its coenzymic function in the diol dehydratase reaction.

A novel analog of adenosylcobalamin in which 5,6-dimethylbenzimidazole and D-ribose moieties of the nucleotide loop are replaced by pyridine and the trimethylene group, respectively, was synthesized and examined for coenzymic function. The coordination of pyridine to the cobalt atom in this analog was stronger than that of 5,6-dimethylbenzimidazole in the corresponding homolog. The adenosyl form of pyridyl analog served as partially active coenzyme for diol dehydratase. The kcat/Km values calculated from the initial velocity indicate that this analog is a better coenzyme than the 5,6-dimethylbenzimidazolyl or imidazolyl counterpart. However, the reaction with the pyridyl analog as coenzyme was accompanied with a concomitant inactivation during catalysis, with a kcat/Kinact value 50-100 times lower than that for adenosylcobalamin or the 5,6-dimethylbenzimidazolyl analog. Therefore, it can be concluded that the 5,6-dimethylbenzimidazole moiety of adenosylcobalamin is important for continuous progress of a catalytic cycle by protecting the reactive intermediates from side reactions.

Benzimidazoles↗
PubMed1994

Microbiological activities of nucleotide loop-modified analogues of vitamin B12.

Novel vitamin B12 analogues in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analogue containing imidazole instead of 5,6-dimethylbenzimidazole as well as cobinamide methyl phosphate were tested for biological activities with Escherichia coli 215, a B12- or methionine-auxotroph, and Lactobacillus leichmannii ATCC 7830 as test organisms. A cyano form of 5,6-dimethylbenzimidazolyl tetramethylene, trimethylene and hexamethylene analogues supported the growth of L. leichmannii in this order. 5,6-Dimethylbenzimidazolyl dimethylene and imidazolyl trimethylene analogues did not show B12 activity and behaved as weak B12 antagonists when added together with cyanocobalamin. An adenosyl form of the biologically active analogues served as coenzymes for ribonucleotide reductase of this bacterium, whereas that of the inactive analogues did not. The latter acted as weak competitive inhibitors against adenosylcobalamin. On the contrary, all the analogues did not support the growth of E. coli 215 at all by themselves and inhibited the growth when added with a suboptimum level of cyanocobalamin. A methyl form of the analogues also did not support the growth of E. coli 215, although they served as active coenzymes for methionine synthase of the bacterium. Since unlabeled analogues strongly inhibited the uptake of [3H]cyanocobalamin by this bacterium, it seems likely that the analogues exert their anti-B12 activity toward E. coli 215 by blocking the B12-transport system.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1993

Adenosylcobinamide methyl phosphate as a pseudocoenzyme for diol dehydrase.

Adenosylcobinamide methyl phosphate, a novel analog of adenosylcobalamin lacking the nucleotide loop moiety, was synthesized. It did not show detectable coenzymic activity but behaved as a strong competitive inhibitor against AdoCbl with relatively high affinity (Ki = 2.5 microM). When apoenzyme was incubated at 37 degrees C with this analog in the presence of substrate, the Co-C bond of the analog was almost completely and irreversibly cleaved within 10 min, forming an enzyme-bound Co(II)-containing species. The cleavage was not observed in the absence of substrate. The Co-C bond cleavage in the presence of substrate was not catalytic but stoichiometric, implying that the Co-C bond of the analog undergoes activation when the analog binds to the active site of the enzyme. 5'-Deoxyadenosine was the only product derived from the adenosyl group of the analog upon the Co-C bond cleavage. Apoenzyme did not undergo modification during this process. Therefore, it seems likely that adenosylcobinamide methyl phosphate acts as a pseudocoenzyme or a potent suicide coenzyme. Since adenosylcobinamide neither functions as coenzyme nor binds tightly to apoenzyme, it can be concluded that the phosphodiester moiety of the nucleotide loop of adenosylcobalamin is essential for tight binding to apoenzyme and therefore for subsequent activation of the Co-C bond and catalysis. It is also evident that the nucleotide loop is obligatory for the normal progress of catalytic cycle.

Apoenzymes↗
PubMed1993

Importance of the nucleotide loop moiety coordinated to the cobalt atom of adenosylcobalamin for coenzymic function in the diol dehydrase reaction.

Three analogs of adenosylcobalamin were synthesized and their coenzymic properties in the diol dehydrase reaction were studied. Neither adenosylcobinamide nor adenosylcobinamide phosphate was active as coenzyme and showed very low affinity for apoenzyme, irrespective of the presence of nucleotide loop fragments, such as 5,6-dimethylbenzimidazole, alpha-D-ribazole, or alpha-D-ribazole-3'-phosphate. The coordination of pyridine to the cobalt atom neither confers the coenzymic function upon adenosylcobinamide nor strengthens the inhibitory effect of cyanoaquacobinamide and methylcobinamide significantly. The analog of adenosylcobalamin in which the N-3 position of 5,6-dimethylbenz-imidazole is methylated was also not active as coenzyme and showed very low affinity for apoenzyme. Since 3,5,6-trimethylbenzimidazole in this analog is no longer coordinated to the cobalt atom, these results show that at least a part of the nucleotide loop moiety coordinated to the cobalt atom of adenosylcobalamin is essential for tight binding to the apoenzyme and therefore for manifestation of coenzymic function.

Binding Sites↗
PubMed1991

Roles of the D-ribose and 5,6-dimethylbenzimidazole moieties of the nucleotide loop of adenosylcobalamin in manifestation of coenzymic function in the diol dehydrase reaction.

Coenzyme analogs in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analog containing imidazole instead of 5,6-dimethylbenzimidazole were synthesized. Coordination of the 5,6-dimethylbenzimidazole to the cobalt atom in these analogs was much weaker than that in cobalamins. The replacement of this base with imidazole did not significantly alter the strength of the coordination to the cobalt atom. 5,6-Dimethylbenzimidazolyl trimethylene and tetramethylene and imidazolyl trimethylene analogs were partially active as coenzymes in the diol dehydrase reaction in this order as judged by kcat, but the others were not active as coenzymes and were weak competitive inhibitors. This indicates that neither the alpha-D-ribofuranose ring nor the functional groups of the ribose moiety are essential for coenzymic function. There was an optimum loop size of the analogs for catalysis and for tight binding to the apoenzyme, which corresponds to the loop size of cobalamins. Therefore, the D-ribose moiety seems important as a spacer to keep the base in the proper position. The reaction with the imidazolyl trimethylene analog as coenzyme was accompanied with concomitant rapid inactivation during catalysis. The inactivation occurred only in the presence of substrate. Upon inactivation with this analog, 5'-deoxyadenosine and a B12r-like species were formed from the adenosyl group and the rest of the analog molecule, respectively, without modification of the apoenzyme. Therefore, it can be concluded that this is a kind of suicide inactivation which occurred from one of the intermediates in the normal catalytic process. The dimethylbenzo moiety of the regular coenzyme thus seems to play an important role in preventing the intermediate complexes from inactivation during catalysis.

Apoenzymes↗
PubMed1990

Essential histidine residues in coenzyme B12-dependent diol dehydrase: dye-sensitized photooxidation and ethoxycarbonylation.

The apoenzyme of diol dehydrase was inactivated by photoirradiation in the presence of rose bengal or methylene blue, following pseudo-first-order kinetics. The inactivation rates were markedly reduced under a helium atmosphere, suggesting that the inactivation is due to photooxidation of the enzyme under air. The half-maximal rate of methylene blue-sensitized photoinactivation was observed at pH around 7.5. Amino acid analyses indicated that one to two histidine residues decreased upon the dye-sensitized photoinactivation, whereas the numbers of tyrosine, methionine, and lysine did not change. Ethoxyformic anhydride, another histidine-modifying reagent, also inactivated diol dehydrase, with pseudo-first-order kinetics and a half-maximal rate at pH 7.7. It was shown spectrophotometrically that three histidine residues per enzyme molecule were modified by this reagent with loss of enzyme activity. Two tyrosine residues per enzyme molecule were also modified rapidly, irrespective of the activity. The photooxidation or ethoxycarbonylation of the enzyme did not result in dissociation of the enzyme into subunits, but deprived the enzyme of ability to bind cyanocobalamin. The percentage loss of cobalamin-binding ability agreed well with the extent of inactivation. The enzyme-bound hydroxocobalamin showed only partial protecting effect against photoinactivation and resulting loss of the cobalamin-binding ability. These results provide evidence that diol dehydrase possesses essential histidine residues which are required for the coenzyme binding.

Amino Acids↗
PubMed1988

Acceleration of cleavage of the carbon-cobalt bond of sterically hindered alkylcobalamins by binding to apoprotein of diol dehydrase.

Cleavage of the C-Co bond of sterically hindered alkylcobalamins bearing neither an adenine moiety nor functional groups, such as isobutylcobalamin, neopentylcobalamin, and cyclohexylcobalamin, was markedly accelerated by their interaction with apoprotein of diol dehydrase, although these cobalamins do not function as coenzyme. Acceleration of the conversion of alkylcobalamins to enzyme-bound hydroxocobalamin was stoichiometric and obeyed first-order reaction kinetics. These results, together with strong competitive inhibition by these alkylcobalamins with respect to adenosylcobalamin, indicate that acceleration of the C-Co bond cleavage by the apoenzyme is due to labilization of their C-Co bond by binding to the active site of the enzyme. This labilization is considered to be caused by a steric distortion of the corrin ring which is induced by specific tight interaction of the cobalamin moiety with apoprotein. The importance of such a labilizing effect for activation of the C-Co bond of adenosylcobalamin in enzymatic reactions is discussed.

Allosteric Site↗