Hemoglobin A 2 (α 2 δ 2), which is present at low concentration (1-2%) in the circulating red cel... more Hemoglobin A 2 (α 2 δ 2), which is present at low concentration (1-2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic. However, reports on its functional properties regarding O 2 binding are conflicting. We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A 2 and systematically studying its functional properties. The construct expressing HbA 2 contains only α and δ genes so that the extensive purification required to isolate natural HbA 2 is circumvented. Although natural hemoglobin A 2 is expressed at low levels in vivo, the amount of recombinant α 2 δ 2 expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the α + β or the α + γ genes, respectively, are present on the construct. Recombinant HbA 2 is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA. However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin. Molecular modeling studies which attempt to understand these properties of HbA 2 are described.
For many years, clinical studies have suggested that blood levels of l-methionine and L-homocyste... more For many years, clinical studies have suggested that blood levels of l-methionine and L-homocysteine correlate with health status or homocystinuria/hypermethioninemia. l-Methionine in a solution containing 0%, 10%, or 20% human serum was detected in 10–200 µM using l-methionine decarboxylase (MetDC). Spike and recovery tests showed that the enzymatic assay could accurately and reproducibly determine the increases in l-methionine in serum samples. These results suggest that our enzymatic method using MetDC is useful for primary screening of hypermethioninemia or homocystinuria based on serum l-methionine concentration. Additionally, we confirmed that l-methionine (100 nmol) in solution was degraded to less than the detection limit by incubation at 37ºC for 10 min using 2 U of MetDC. Therefore, l-homocysteine in serum samples can be detected with equivalent sensitivity using l-methionine γ-lyase (MGL), in solutions that either did not contain l-methionine or contained l-methionine pre...
Acta crystallographica. Section F, Structural biology communications, Mar 1, 2017
Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)-dependent enzyme,... more Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an L-homoserine derivative and L-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P21; unit-cell parameters a = 58.4, b = 149.3, c = 90.2 Å, β = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C2221; unit-cell paramet...
Bioscience, biotechnology, and biochemistry, Jan 13, 2016
Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor... more Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-(14)C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The KM and kcat for Sephs2-Sec60Cys were determined to be 26 μM and 0.352 min(-1), respectively.
Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high... more Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni-Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.
A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibi... more A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD.
2-Nitropropane dioxygenase, purified to homogeneity by an improved method from a yeast, Hansenula... more 2-Nitropropane dioxygenase, purified to homogeneity by an improved method from a yeast, Hansenula mrakii, has a molecular weight of 42,000, and consists of a single polypeptide. The enzyme contains I mol of FAD per mol of enzyme. The iron protein associated with previous preparations was removed by the present purification procedures. The enzyme catalyzes the oxygenative denitrification of anionic nitroalkanes much more effectively than that of the neutral ones with the optimum pH of 6.5. The Michaelis constants for the anionic substrates are as follows: 2-nitropropane, 1.61 mM; I-nitropropane, 3.23 mM; nitroethane, 3.13 mM, and 3-nitro-2-butanol, 0.59mM.
We have determined the x-ray crystal structure of L-lysine ε-oxidase from Marinomonas mediterrane... more We have determined the x-ray crystal structure of L-lysine ε-oxidase from Marinomonas mediterranea in its native and L-lysine-complex forms at 1.94- and 1.99-Å resolution, respectively. In the native enzyme, electron densities clearly indicate the presence of cysteine tryptophylquinone (CTQ) previously identified in quinohemoprotein amine dehydrogenase. In the L-lysine-complex, an electron density corresponding to the bound L-lysine shows that its ε-amino group is attached to the C6 carbonyl group of CTQ, suggesting the formation of a Schiff-base intermediate. Collectively, the present crystal structure provides the first example of an enzyme employing a tryptophylquinone cofactor in an amine oxidase.
Lovastatin production is dependent on the substrates provided. We investigated how several carbon... more Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose-glycerol-peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.
The regulatory region of Thiobacillus ferrooxidans leuB gene was analyzed. The deletion analysis ... more The regulatory region of Thiobacillus ferrooxidans leuB gene was analyzed. The deletion analysis indicated that the promoter sequences CA TCCG and T A TT AT, which were similar to the consensus-35 and-10 sequences of Escherichia coli, respectively, existed. The transcriptional initiation site was located at the position of cytosine-26. The deletion analysis of the upstream region suggested the existence of a regulatory region by leucine and the region related to transcription of the gene.
The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, ... more The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR30 1, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of M , of 39 000. At the optimum temperature (50 "C), the enzyme has a specific activity of 1800 units/mg (V,,,, D-to L-alanine). Resolution and reconsititution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme. A l a n i n e racemases (EC 5.1.1.1) catalyze the interconversion
An alanine racemase encoded by a gene from the thermophilic Gram-positive bacterium Bacillus stea... more An alanine racemase encoded by a gene from the thermophilic Gram-positive bacterium Bacillus stearothermophilus is overproduced to 0.3% of the soluble protein when carried on plasmid pICR4 in Escherichia coli [Inagaki, K.
Protein science : a publication of the Protein Society, 2017
Methionine γ-lyse (MGL) catalyzes the α, γ-elimination of l-methionine and its derivatives as wel... more Methionine γ-lyse (MGL) catalyzes the α, γ-elimination of l-methionine and its derivatives as well as the α, β-elimination of l-cysteine and its derivatives to produce α-keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site-directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51-Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5'-phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with t...
In anaerobic or aerobic conditions in the presence of 5 mM sodium cyanide, an inhibitor of iron o... more In anaerobic or aerobic conditions in the presence of 5 mM sodium cyanide, an inhibitor of iron oxidase, cupric ion (Cu2+) was reduced enzymatically with elemental sulfur (SO) by washed intact cells of ThiobaciUus ferrooxidans AP19-3 to give cuprous ion (Cu+). The rate of Cu2' reduction was proportional to the concentrations of So and Cu2' added to the reaction mixture. The pH optimum for the cupric ion-reducing system was 5.0, and the activity was completely destroyed by 10-min incubation of cells at 70TC. The activity of Cu2' reduction with S°by this strain was strongly inhibited by inhibitors of hydrogen sulfide: ferric ion oxidoreductase (SFORase), such as a,a'-dipyridyl, 4,5-dihydroxy-m-benzene disulfonic acid disodium salts, and diazine dicarboxylic acid bis-(N, N-dimethylamide). A SFORase purified from this strain, which catalyzes oxidation of both hydrogen sulfide and S°with Fe3+ or Mo6+ as an electron acceptor in the presence of glutathione, catalyzed a reduction of Cu2' by So, and the Michaelis constant of SFORase for Cu2+ was 7.2 mM, indicating that a SFORase catalyzes the reduction of not only Fe3+ and Mo6+ but also Cu2+.
L-Methionine γ-lyase is a pyridoxal 5′-phosphatedependent enzyme which has tumor selective antica... more L-Methionine γ-lyase is a pyridoxal 5′-phosphatedependent enzyme which has tumor selective anticancer activity. An efficient production process for the recombinant enzyme was constructed by using the overexpression plasmid in Escherichia coli, large-scale cultivation, and practical crystallization on an industrial scale. The plasmid was optimized with a promoter and the region of the ribosome-binding site. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The transformants produced the enzyme, which intracellularly accumulated at 2.1 mg/ml as an active form and accounted for 43% of the total proteins in the soluble fraction by simple batch fermentation using a 500-l fermentor. The crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100-l crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. We prepared 600 g of purified enzyme with a low endotoxin content of sufficient quality for therapeutical use, with a 41% overall yield in the purification process.
Hemoglobin A 2 (α 2 δ 2), which is present at low concentration (1-2%) in the circulating red cel... more Hemoglobin A 2 (α 2 δ 2), which is present at low concentration (1-2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic. However, reports on its functional properties regarding O 2 binding are conflicting. We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A 2 and systematically studying its functional properties. The construct expressing HbA 2 contains only α and δ genes so that the extensive purification required to isolate natural HbA 2 is circumvented. Although natural hemoglobin A 2 is expressed at low levels in vivo, the amount of recombinant α 2 δ 2 expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the α + β or the α + γ genes, respectively, are present on the construct. Recombinant HbA 2 is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA. However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin. Molecular modeling studies which attempt to understand these properties of HbA 2 are described.
For many years, clinical studies have suggested that blood levels of l-methionine and L-homocyste... more For many years, clinical studies have suggested that blood levels of l-methionine and L-homocysteine correlate with health status or homocystinuria/hypermethioninemia. l-Methionine in a solution containing 0%, 10%, or 20% human serum was detected in 10–200 µM using l-methionine decarboxylase (MetDC). Spike and recovery tests showed that the enzymatic assay could accurately and reproducibly determine the increases in l-methionine in serum samples. These results suggest that our enzymatic method using MetDC is useful for primary screening of hypermethioninemia or homocystinuria based on serum l-methionine concentration. Additionally, we confirmed that l-methionine (100 nmol) in solution was degraded to less than the detection limit by incubation at 37ºC for 10 min using 2 U of MetDC. Therefore, l-homocysteine in serum samples can be detected with equivalent sensitivity using l-methionine γ-lyase (MGL), in solutions that either did not contain l-methionine or contained l-methionine pre...
Acta crystallographica. Section F, Structural biology communications, Mar 1, 2017
Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)-dependent enzyme,... more Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an L-homoserine derivative and L-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P21; unit-cell parameters a = 58.4, b = 149.3, c = 90.2 Å, β = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C2221; unit-cell paramet...
Bioscience, biotechnology, and biochemistry, Jan 13, 2016
Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor... more Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-(14)C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The KM and kcat for Sephs2-Sec60Cys were determined to be 26 μM and 0.352 min(-1), respectively.
Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high... more Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni-Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.
A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibi... more A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD.
2-Nitropropane dioxygenase, purified to homogeneity by an improved method from a yeast, Hansenula... more 2-Nitropropane dioxygenase, purified to homogeneity by an improved method from a yeast, Hansenula mrakii, has a molecular weight of 42,000, and consists of a single polypeptide. The enzyme contains I mol of FAD per mol of enzyme. The iron protein associated with previous preparations was removed by the present purification procedures. The enzyme catalyzes the oxygenative denitrification of anionic nitroalkanes much more effectively than that of the neutral ones with the optimum pH of 6.5. The Michaelis constants for the anionic substrates are as follows: 2-nitropropane, 1.61 mM; I-nitropropane, 3.23 mM; nitroethane, 3.13 mM, and 3-nitro-2-butanol, 0.59mM.
We have determined the x-ray crystal structure of L-lysine ε-oxidase from Marinomonas mediterrane... more We have determined the x-ray crystal structure of L-lysine ε-oxidase from Marinomonas mediterranea in its native and L-lysine-complex forms at 1.94- and 1.99-Å resolution, respectively. In the native enzyme, electron densities clearly indicate the presence of cysteine tryptophylquinone (CTQ) previously identified in quinohemoprotein amine dehydrogenase. In the L-lysine-complex, an electron density corresponding to the bound L-lysine shows that its ε-amino group is attached to the C6 carbonyl group of CTQ, suggesting the formation of a Schiff-base intermediate. Collectively, the present crystal structure provides the first example of an enzyme employing a tryptophylquinone cofactor in an amine oxidase.
Lovastatin production is dependent on the substrates provided. We investigated how several carbon... more Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose-glycerol-peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.
The regulatory region of Thiobacillus ferrooxidans leuB gene was analyzed. The deletion analysis ... more The regulatory region of Thiobacillus ferrooxidans leuB gene was analyzed. The deletion analysis indicated that the promoter sequences CA TCCG and T A TT AT, which were similar to the consensus-35 and-10 sequences of Escherichia coli, respectively, existed. The transcriptional initiation site was located at the position of cytosine-26. The deletion analysis of the upstream region suggested the existence of a regulatory region by leucine and the region related to transcription of the gene.
The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, ... more The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR30 1, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of M , of 39 000. At the optimum temperature (50 "C), the enzyme has a specific activity of 1800 units/mg (V,,,, D-to L-alanine). Resolution and reconsititution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme. A l a n i n e racemases (EC 5.1.1.1) catalyze the interconversion
An alanine racemase encoded by a gene from the thermophilic Gram-positive bacterium Bacillus stea... more An alanine racemase encoded by a gene from the thermophilic Gram-positive bacterium Bacillus stearothermophilus is overproduced to 0.3% of the soluble protein when carried on plasmid pICR4 in Escherichia coli [Inagaki, K.
Protein science : a publication of the Protein Society, 2017
Methionine γ-lyse (MGL) catalyzes the α, γ-elimination of l-methionine and its derivatives as wel... more Methionine γ-lyse (MGL) catalyzes the α, γ-elimination of l-methionine and its derivatives as well as the α, β-elimination of l-cysteine and its derivatives to produce α-keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site-directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51-Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5'-phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with t...
In anaerobic or aerobic conditions in the presence of 5 mM sodium cyanide, an inhibitor of iron o... more In anaerobic or aerobic conditions in the presence of 5 mM sodium cyanide, an inhibitor of iron oxidase, cupric ion (Cu2+) was reduced enzymatically with elemental sulfur (SO) by washed intact cells of ThiobaciUus ferrooxidans AP19-3 to give cuprous ion (Cu+). The rate of Cu2' reduction was proportional to the concentrations of So and Cu2' added to the reaction mixture. The pH optimum for the cupric ion-reducing system was 5.0, and the activity was completely destroyed by 10-min incubation of cells at 70TC. The activity of Cu2' reduction with S°by this strain was strongly inhibited by inhibitors of hydrogen sulfide: ferric ion oxidoreductase (SFORase), such as a,a'-dipyridyl, 4,5-dihydroxy-m-benzene disulfonic acid disodium salts, and diazine dicarboxylic acid bis-(N, N-dimethylamide). A SFORase purified from this strain, which catalyzes oxidation of both hydrogen sulfide and S°with Fe3+ or Mo6+ as an electron acceptor in the presence of glutathione, catalyzed a reduction of Cu2' by So, and the Michaelis constant of SFORase for Cu2+ was 7.2 mM, indicating that a SFORase catalyzes the reduction of not only Fe3+ and Mo6+ but also Cu2+.
L-Methionine γ-lyase is a pyridoxal 5′-phosphatedependent enzyme which has tumor selective antica... more L-Methionine γ-lyase is a pyridoxal 5′-phosphatedependent enzyme which has tumor selective anticancer activity. An efficient production process for the recombinant enzyme was constructed by using the overexpression plasmid in Escherichia coli, large-scale cultivation, and practical crystallization on an industrial scale. The plasmid was optimized with a promoter and the region of the ribosome-binding site. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The transformants produced the enzyme, which intracellularly accumulated at 2.1 mg/ml as an active form and accounted for 43% of the total proteins in the soluble fraction by simple batch fermentation using a 500-l fermentor. The crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100-l crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. We prepared 600 g of purified enzyme with a low endotoxin content of sufficient quality for therapeutical use, with a 41% overall yield in the purification process.
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Papers by Kenji Inagaki