Search PubMed⌕ Search

Biomedical subjects

Masatoshi Goto

Publications and source records attributed to Masatoshi Goto.

At least 19 recordsLinked to original sources
PubMed2006

Steady-state kinetic characterization of evolved biphenyl dioxygenase, which acquired novel degradation ability for benzene and toluene.

Biphenyl dioxygenase (Bph Dox) catalyzes initial oxygenation in the bacterial biphenyl degradation pathway. Bph Dox in Pseudomonas pseudoalcaligenes KF707 is a Rieske type three-component enzyme in which a large subunit (encoded by the bphA1 gene) plays an important role in the substrate specificity of Bph Dox. Steady-state kinetic assays using purified enzyme components demonstrated that KF707 Bph Dox had a kcat/Km of 33.1 x 10(3) (M(-1) s(-1)) for biphenyl. Evolved 1072 Bph Dox generated by the process of DNA shuffling (Suenaga, H. et al., J. Bacteriol., 184, 3682-3688 (2002)) exhibited enhanced degradation activity not only for biphenyl (kcat/Km of 62.2 x 10(3) [M(-1) s(-1)]) but also for benzene and toluene, compounds that are rarely attacked by KF707 Bph Dox. These results suggest that evolved 1072 Bph Dox acquires higher affinities and catalytic efficiencies for various substrates than the original KF707 enzyme.

Benzene↗
PubMed2005

Active-site engineering of biphenyl dioxygenase: effect of substituted amino acids on substrate specificity and regiospecificity.

Biphenyl dioxygenase (Bph Dox) catalyzes the initial dioxygenation step in the metabolism of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Previously, the substitution of Asn at Thr-376 near the active-site iron in the BphA1 of Pseudomonas pseudoalcaligenes KF707 expanded the oxidation range and altered the regiospecificity of Bph Dox for PCBs. In this study, we replaced Thr-376 with Gly, Ser, Gln, Tyr, Val, Phe, Asp, and Lys and expressed these enzymes in Escherichia coli. Bph Dox mutants of Thr376Asn, Thr376Val, Thr376Phe, and Thr376Lys showed novel degradation activity for dibenzofuran, which is a poor substrate for KF707 Bph Dox. All active Bph Dox mutants showed altered regiospecificity with 2,2'-dichlorobiphenyl and 2,5,4'-trichlorobiphenyl. The Thr376Gly, Thr376Val, Thr376Phe, and Thr376Asp Bph Dox mutants introduced molecular oxygen at the 2,3 position of 2,2'-dichlorobiphenyl, forming 2-chloro-2',3'-dihydroxybiphenyl with concomitant dechlorination. The Bph Dox mutants of Thr376Gly, Thr376Ser, Thr376Asp, and Thr376Lys attacked 2,5,4'-trichlorobiphenyl via both 2',3'- and 3,4-dioxygenation activities. In particular, the Thr376Phe Bph Dox mutant exhibited enhanced and expanded degradation activities toward all of the compounds tested. Further site-directed mutation was induced to change the oxidizing character of KF707 Bph Dox to that of the Bph Dox of Burkholderia xenovorans LB400 by the substitution of two amino acids, Ile335Phe and Thr376Asn, near the active-site.

Amino Acid Substitution↗
PubMed2005

Emergence of two types of nondechlorinating variants in the tetrachloroethene-halorespiring Desulfitobacterium sp. strain Y51.

Desulfitobacterium sp. strain Y51 exhibits a strong dechlorinating activity for tetrachloroethene (PCE), converting it to cis-1,2-dichloroethene via trichloroethene by the action of the PceA reductive dehalogenase (encoded by pceA). The gene organization around the pceA gene cluster was determined to be in the following order: orf4, orf3, ISDesp1, pceA-B-C-T-mcpA, and ISDesp2, where the pceA gene cluster is surrounded by two nearly identical copies of the ISDesp insertion sequence. Serial subculture of strain Y51 gave rise to variants that abolished the PCE-dechlorination activity. Southern hybridization analysis revealed two types of variants termed small deletion (SD) and large deletion (LD). The characterization of both variants revealed a genetic rearrangement around the pceAB gene cluster. In variant SD, ISDesp1 comprised of 1,572 bp was deleted, which includes the tnpAa encoding IS256 family transposase and unknown orf1. The ISDesp1 contained the inverted terminal repeat sequence and a -35 promoter stretch just upstream of the pceA gene, indicating that this IS element is involved in the formation of the variant SD. Loss of the pceA transcription changed the variant SD to the PCE-nondechlorinating phenotype. The variant LD lost the 6.5-kb region, including one copy of ISDesp and the pceABCT-mcpA gene cluster, confirming that the homologous recombination is associated with the emergence of this variant.

Base Sequence↗
PubMed2005

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review.

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 microM and as low as 0.06 microM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min(-1) mg protein(-1). The apparent K(m) values for PCE and TCE were 105.7 and 535.3 microM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.

Amino Acid Sequence↗
PubMed2005

Protein O-mannosyltransferase A of Aspergillus awamori is involved in O-mannosylation of glucoamylase I.

Industrially important extracellular enzymes from filamentous fungi are often O-mannosylated. The structure and function of the pmtA (AapmtA) gene encoding the protein O-D-mannosyltransferase of Aspergillus awamori were characterized. The AapmtA disruptant, designated AaPMTA, was constructed by homologous recombination. The strain AaPMTA exhibited fragile cell morphology with respect to hyphal extension, as well as swollen hyphae formation and conidia formation in potato dextrose medium. Moreover, the AapmtA disruptant showed increased sensitivity to high temperature and Congo red. Thus, the AaPmtA protein is involved in the formation of the normal cell wall. The strain AaPMTA could grow well in liquid synthetic medium and secrete glucoamylase I (GAI-AaPMTA) to a similar extent to the wild-type strain (GAI-WT). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the GAIs revealed that approximately 33 mannose moieties of GAI were absent in strain AaPMTA. This result indicates that the AaPmtA protein is responsible for the transfer of mannose to GAI. Structural analysis of the O-linked oligosaccharides of GAI also demonstrated that the AapmtA disruption resulted in a reduction of the amounts of O-linked oligosaccharides, such as D-mannose and alpha-1,2-mannotriose, in GAI-AaPMTA. However, the amount of alpha-1,2-mannobiose was comparable between GAI-WT and GAI-AaPMTA. The result suggests the presence of a compensatory mechanism in the synthetic pathway of O-mannosylation in A. awamori.

Aspergillus↗
PubMed2004

Modulation of mammalian cell growth and death by prokaryotic and eukaryotic cytochrome c.

Cytochrome c(551), an 8,685-Da haem-containing protein, is known to be involved in electron transfer during dissimilative denitrification by Pseudomonas aeruginosa. Both cytochrome c(551) and copper-containing redox protein azurin have been used in vitro as partners in electron transfer. Azurin has been reported to induce apoptosis in macrophages and cancer cells. We now report that, unlike azurin, cytochrome c(551), termed Cyt c(551), has very little ability to induce apoptosis in J774 cell line-derived macrophages but demonstrates significant inhibition of cell cycle progression in such cells. A mutant form of Cyt c(551), V23DI59E, has significantly reduced ability to inhibit cell cycle progression but demonstrates a higher level of apoptosis-inducing activity in macrophages, compared with WT Cyt c(551). Interestingly, the WT Cyt c(551), but not the mutant form, significantly enhances the level of tumor suppressor protein p16(Ink4a), a known inhibitor of cell cycle progression whereas the mutant form seems to form a complex with tumor suppressor protein p53, thereby enhancing its intracellular level to some extent. Eukaryotic cytochromes such as horse and bovine cytochrome c have also been shown to induce apoptosis but not inhibition of cell cycle progression in J774 cells, thus signifying a role of this redox protein in entry to, and in the induction of, cell death in mammalian cells.

Amino Acid Sequence↗
PubMed2004

Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans.

Protein O-glycosylation is essential for protein modification and plays important roles in eukaryotic cells. O-Mannosylation of proteins occurs in the filamentous fungus Aspergillus. The structure and function of the pmtA gene, encoding protein O-d-mannosyltransferase, which is responsible for the initial O-mannosylation reaction in Aspergillus nidulans, was characterized. Disruption of the pmtA gene resulted in the reduction of in vitro protein O-d-mannosyltransferase activity to 6 % of that of the wild-type strain and led to underglycosylation of an extracellular glucoamylase. The pmtA disruptant exhibited abnormal cell morphology and alteration in carbohydrate composition, particularly reduction in the skeletal polysaccharides in the cell wall. The results indicate that PmtA is required for the formation of a normal cell wall in A. nidulans.

Aspergillus nidulans↗
PubMed2004

Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase.

We engineered biphenyl-degrading Alcaligenes sp. strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707). Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination. The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions. The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions. Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.

Alcaligenes↗
PubMed2004

Thr/Ser-rich domain of Aspergillus glucoamylase is essential for secretion.

The recombinant Aspergillus awamori strain carrying the mutant glucoamylase-encoding gene in which the entire Thr/Ser-rich Gp-I domain was deleted abolished secretion of mutant glucoamylase. The transcription of the Bip-encoding bipA was low in the wild type (wt) strain, but elevated in the recombinant strain under the condition of glaA expression. The results indicate that the Gp-I domain is vital for glucoamylase secretion.

Aspergillus↗
PubMed2003

Induction of apoptosis in macrophages by Pseudomonas aeruginosa azurin: tumour-suppressor protein p53 and reactive oxygen species, but not redox activity, as critical elements in cytotoxicity.

Azurin is a copper-containing protein involved in electron transfer during denitrification. We reported recently that purified azurin demonstrates cytotoxicity to macrophages by forming a complex with the tumour-suppressor protein p53, thereby stabilizing it and enhancing its function as an inducer of proapoptotic activity (Yamada, T., Goto, M., Punj, V., Zaborina, O., Kimbara, K., Das Gupta, T. K., and Chakrabarty, A. M. 2002, Infect Immun70: 7054-7062). It is, however, not known whether the oxidoreductase (redox) activity of azurin or the involvement of copper is important for its cytotoxicity. We have isolated apo-azurin devoid of copper and site-directed mutants that are redox negative because of either replacement of a cysteine residue (Cys-112) involved in co-ordination with copper or mutational replacement of two methionine residues (Met-44 and Met-64) that are present in the hydrophobic patch of azurin and allow interaction of azurin with its redox partner cytochrome c551. We demonstrate that, although the wild type (wt) and the Cys-112 Asp mutant azurin can form complexes with the tumour-suppressor protein p53 and generate high levels of reactive oxygen species (ROS), the redox-negative Met-44LysMet-64Glu mutant azurin is defective in complex formation with p53, generates low levels of ROS and lacks appreciable cytotoxicity towards macrophages. Thus, complex formation with p53 and ROS generation, rather than azurin redox activity, are important in the cytotoxic action of azurin towards macrophages.

Amino Acid Sequence↗
PubMed2003

[Not Available].

Explore the source record for details and available documents.

Journal Article↗
PubMed2002

Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer.

The use of live bacteria in the treatment of cancer has a long and interesting history. We report the use of a purified bacterial redox protein, azurin, that enters human cancer (melanoma UISO-Mel-2) cells and induces apoptosis. The induction of apoptosis occurs readily in melanoma cells harboring a functional tumor suppressor protein p53, but much less efficiently in p53-null mutant melanoma (UISO-Mel-6) cells. A redox-negative mutant form of azurin (M44K/M64E) demonstrates much less cytotoxicity to the UISO-Mel-2 cells than the wild-type protein. Azurin has been shown to be internalized in UISO-Mel-2 cells and is localized predominantly in the cytosol and in the nuclear fraction. In the p53-null UISO-Mel-6 cells, azurin is localized only in the cytosol. Thus, intracellular trafficking of azurin to the nucleus is p53-dependent. Azurin forms a complex with p53, thereby stabilizing it and raising its intracellular level in cytosolic, mitochondrial, and nuclear fractions. Corresponding to an increasing level of p53, an inducer of apoptosis, the level of Bax also increases in mitochondria, allowing significant release of mitochondrial cytochrome c into the cytosol, thus initiating the onset of apoptosis. The M44K/M64E mutant form of azurin, deficient in cytotoxicity, is also deficient in forming a complex with p53 and is less efficient in stabilizing p53 than wild-type azurin. Azurin has been shown to allow regression of human UISO-Mel-2 tumors xenotransplanted in nude mice and may potentially be used in cancer treatment.

Animals↗
PubMed2002

Molecular breeding of Aspergillus kawachii overproducing cellulase and its application to brewing barley shochu.

In order to improve fermentation of barley without addition of commercial cellulase, a white koji mold, Aspergillus kawachii IFO4308, was transformed with the egl1 gene encoding endoglucanase I (EGI) of Trichoderma viride and the endogenous cekA gene encoding endoglucanase (CekA). Transformants with egl1 under the control of the strong glaA promoter produced EGI in both submerged and solid-state cultures. However, the EGI produced in solid-state culture was unstable due to the acidic condition of this culture. A transformant N10 with two additional copies of the cekA gene exhibited endoglucanase activities against carboxymethyl-cellulose, which are 21- and 1.8-fold higher than that of the wild-type (wt) strain when the cells were cultivated in submerged and solid-state cultures, respectively. Cultivation of strain N10 in steamed barley for preparing koji followed by fermentation with Saccharomyces cerevisiae resulted in improved fermentation assessed based on higher productions of ethanol, amino acids, and organic acids, the reduction of residual sugar, and the low viscosity of barley mash. The overall fermentation result for the transformant carrying cekA was comparable with that for the wt strain using commercial cellulase. These results demonstrate that acquisition of only two-fold CekA activity by A. kawachii in the solid-state culture allows us to improve the brewing of barley shochu.

Journal Article↗
PubMed2002

Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase.

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the delta-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. delta-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.

Chromosomes, Fungal↗
PubMed2002

The bacterial redox protein azurin induces apoptosis in J774 macrophages through complex formation and stabilization of the tumor suppressor protein p53.

Two redox proteins, azurin and cytochrome c(551) elaborated by Pseudomonas aeruginosa, demonstrate significant cytotoxic activity towards macrophages. Azurin can enter macrophages, localize in the cytosol and nuclear fractions, and induce apoptosis. Two redox-negative mutants of azurin have less cytotoxicity than does wild-type (wt) azurin. Azurin has been shown to form a complex with the tumor suppressor protein p53, a known inducer of apoptosis, thereby stabilizing it and enhancing its intracellular level. A higher level of reactive oxygen species (ROS), generated during treatment of macrophages with wt azurin, correlates with its cytotoxicity. Treatment with some ROS-removing antioxidants greatly reduces azurin-mediated cytotoxicity, thus demonstrating a novel virulence property of this bacterial redox protein.

Animals↗
PubMed2002

Effects of amino acid alterations on the transglycosylation reaction of endoglucanase I from Trichoderma viride HK-75.

Endoglucanase I (EGI) from Trichoderma viride HK-75 catalyzes not only hydrolysis but also transglycosylation reactions of cellooligosaccharides. In order to characterize the important amino acid residues in transglycosylation of EGI, three Tyr, one Leu, and two Glu residues of EGI were replaced by Trp or Asp. The seven resulting EGI, except for L200W, had reduced activities toward carboxymethyl-cellulose compared to that of wild type EGI. The results from the mutations in the catalytic residues of E196 and E201 indicate that the space just around the catalytic residues is not directly related to the transglycosylation reactions of EGI. Analyses of the enzymes with mutations in the substrate-binding residues showed that Y146, Y170, and L200 of EGI are closely involved in the mode of transglycosylation and that several amino acid residues within the active site are involved in the transglycosylation reaction of EGI.

Amino Acid Substitution↗