Search PubMed⌕ Search

Biomedical subjects

S Horinouchi

Publications and source records attributed to S Horinouchi.

At least 91 records · Page 5Linked to original sources
PubMed1996

The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2).

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essential for aerial mycelium formation and streptomycin (Sm) production in Streptomyces griseus. A protein Ser/Thr kinase (AfsK), the product of the Streptomyces coelicolor A3(2) afsK gene, controlling secondary metabolism in this strain, reversed the aerial mycelium-negative phenotype of an A-factor-deficient mutant strain, S. griseus HH1, and induced sporulation without affecting A-factor productivity or Sm production. A mutant AfsK protein lacking kinase activity failed to induce aerial mycelium formation which indicates the importance of the kinase activity for suppression in S. griseus. These data suggest that a Ser/Thr kinase functionally similar to S. coelicolor A3(2) AfsK plays a regulatory role in aerial mycelium formation in S. griseus, either as a member in the A-factor regulatory network or independently of this network.

4-Butyrolactone↗
PubMed1996

Integrative transformation of the zygomycete Rhizomucor pusillus by homologous recombination.

For development of a homologous transformation system for the zygomycete fungus, Rhizomucor pusillus, the isopropylmalate isomerase (leuA) gene was cloned from R. pusillus IFO 4578 by the DNA-probing method with the leuA sequence of Mucor circinelloides as probe. The nucleotide sequence revealed that leuA of R. pusillus encoded a 755-amino-acid protein of 82.5 kDa with no intron. The leuA gene on pUC19 (plasmid pRPLeu10) was introduced by polyethyleneglycol-assisted transformation into protoplasts of a leuA- mutant of R. pusillus that was obtained by UV mutagenesis. Transformation under optimal conditions yielded 20 Leu+ transformants (micrograms pRPLeu10 DNA)-1 (1 x 10(6) viable protoplasts)-1. Blot analysis of DNA from the transformants showed that the pRPLeu10 sequence was integrated into the genome by homologous recombination at the leuA locus.

Amino Acid Sequence↗
PubMed1996

Involvement of a residue at position 75 in the catalytic mechanism of a fungal aspartic proteinase, Rhizomucor pusillus pepsin. Replacement of tyrosine 75 on the flap by asparagine enhances catalytic efficiency.

Residue 75 on the flap, a beta hairpin loop that partially covers the active site cleft, is tyrosine in most members of the aspartic proteinase family. Site-directed mutagenesis was carried out to investigate the functional role of this residue in Rhizomucor pusillus pepsin, an aspartic proteinase with high milk-clotting activity produced by the fungus Rhizomucor pusillus. A set of mutated enzymes with replacement of the amino acid at position 75 by 17 other amino acid residues except for His and Gly was constructed and their enzymatic properties were examined. Strong activity, higher than that of the wild-type enzyme, was found in the mutant with asparagine (Tyr75Asn), while weak but distinct activity was observed in Tyr75Phe. All the other mutants showed markedly decreased or negligible activity, less than 1/1000 of that of the wild-type enzyme. Kinetic analysis of Tyr75Asn using a chromogenic synthetic oligopeptide as a substrate revealed a marked increase in kcat with slight change in K(m), resulting in a 5.6-fold increase in kcat/K(m). When differential absorption spectra upon addition of pepstatin, a specific inhibitor for aspartic proteinase, were compared between the wild-type and mutant enzymes, the wild-type enzyme and Tyr75Asn, showing strong activity, had spectra with absorption maxima at 280, 287 and 293 nm, whereas the others, showing decreased or negligible activity, had spectra with only two maxima at 282 and 288 nm. This suggests a different mode of the inhibitor binding in the latter mutants. These observations suggest a crucial role of the residue at position 75 in enhancing the catalytic efficiency through affecting the mode of substrate-binding in the aspartic proteinases.

Amino Acid Sequence↗
PubMed1996

Conversion of a cyanhydrin compound into S-(-)-3-phenyllactic acid by enantioselective hydrolytic activity of Pseudomonas sp. BC-18.

A Pseudomonas strain, named BC-18, which can convert racemic phenylacetaldehyde-cyanhydrin (3-phenyllactonitrile) enantioselectively to S-(-)-3-phenyllactic acid (S-PLA), was isolated from soil. Although PLA produced with intact cells contained the S enantiomers of approximately 75% enantiomeric excess (% e.e.), repeated crystallization gave a higher purity (99.8% e.e.) of the S configuration product. Production of S-PLA was significantly increased when 2.0% (w/v) of calcium chloride were added to the reaction mixture for precipitation of S-PLA. Chemical mutagenesis yielded a mutant strain, named BC348-9, with 16 times higher activity (40 mU/OD630), compared with that of the parent strain (2.5 mU/OD630). When the mutant strain BC348-9 was used, approximately 18 g/OD630 was produced, which is 12 times higher than that of the parent strain. The final accumulation of PLA exceeded 6.0%, 1.2 times higher than that of the parent strain.

Calcium Chloride↗
PubMed1996

Extracellular production of a Serratia marcescens serine protease in Escherichia coli.

The Serratia marcescens serine protease (SSP) is one of the extracellular enzymes secreted from this Gram-negative bacterium. When the ssp gene, which encodes a SSP precursor (preproSSP) composed of a typical NH2-terminal signal peptide, a mature enzyme domain, and a large COOH-terminal pro-region, is expressed in Escherichia coli, the mature protease is excreted through the outer membrane into the medium. The COOH-terminal pro-region, which is integrated into the outer membrane, provides the essential function for the export of the mature protein across the outer membrane. This is a very simple pathway, in contrast to the general secretory pathway exemplified by the secretion of a pullulanase from Klebsiella oxytoca, in which many separately encoded accessory proteins are required for the transport through the outer membrane. Moreover, the NH2-terminal region of 71 amino acid residues of the COOH-terminal pro-sequence plays an essential role, as an "intramolecular chaperone," in the folding of the mature enzyme in the medium. In addition to ssp, the S. marcescens strain contains two ssp homologues encoding proteins similar to SSP in amino acid sequence and size, but with no protease activity. Characterization of the homologue proteins and chimeric proteins between the homologues and SSP, all of which are produced in E. coli, has shown that they are membrane proteins that are localized in the outer membrane in the same manner as for SSP. By use of the COOH-terminal domain of SSP, pseudoazurin was exported to the cell surface of E. coli, which proves the usefulness of the SSP secretory system in the export of foreign proteins across the outer membrane.

Biological Transport↗
PubMed1995

Structure of Alcaligenes faecalis nitrite reductase and a copper site mutant, M150E, that contains zinc.

The structures at 2.0 and 2.25 A resolution of native and recombinant nitrite reductase from Alcaligenes faecalis show that they are identical to each other and very similar to nitrite reductase from Achromobacter cycloclastes. The crystallographic structure of a mutant, M150E, which unlike the wild-type protein cannot be reduced by pseudoazurin, shows that the glutamate replacement for methionine binds to a metal at the type I Cu site via only one oxygen. Anomalous scattering data collected at wavelengths of 1.040 and 1.377 A reveal that the metal at the type I site is a Zn. No significant differences from the native structure other than local perturbations at the type I site are seen. A local pseudo 2-fold axis relates the two domains of different monomers which form the active site. The two residues, Asp98 and His255, believed to be involved in catalysis are related by this 2-fold. An unusual (+)-(+) charge interaction between Lys269, Glu279, and His100 helps to orient the active site Cu ligand, His100. A number of negatively charged surface residues create an electrostatic field whose shape suggests that it may serve to direct incoming negatively charged nitrite as well as to dock the electron donor partner, pseudoazurin.

Alcaligenes↗
PubMed1995

A K-252a-resistance gene, sks1+, encodes a protein similar to the Caenorhabditis elegans F37 A4.5 gene product and confers multidrug resistance in Schizosaccharomyces pombe.

A gene named sks1+ was cloned as a suppressor of the K-252a-sensitivity phenotype of Schizosaccharomyces pombe (Sp) from a gene library of the parental Sp chromosomal DNA constructed with a multicopy vector pDB248'. The gene encoded a 308-amino-acid (aa) protein similar to the Caenorhabditis elegans F37 A4.5 gene product and to the mouse and Drosophila Mov34 gene products. The sks1+ null mutants obtained by gene disruption were non-viable, indicating that sks1+ is essential for vegetative growth. The parental Sp strain carrying multiple copies of sks1+ showed distinct cross-resistance to staurosporine, thiabendazole and vanadate in addition to K-252a, although Sks1 has no similarity in aa sequence to those of ATP-binding cassette (ABC)-type transporters. The multicopy plasmid containing sks1+ conferred multidrug resistance (MDR), even in a mutant cell defective in pmd1+ encoding an ABC-type transporter. It is therefore unlikely that the function of pmd1+ is involved in MDR conferred by sks1+. These results suggest that sks1+ is a functionally novel MDR gene.

Amino Acid Sequence↗
PubMed1995

Trichostatin A and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function.

Reversible acetylation at the epsilon-amino group of lysines located at the conserved domain of core histones is supposed to play an important role in the regulation of chromatin structure and its transcriptional activity. One promising strategy for analyzing the precise function of histone acetylation is to block the activities of acetylating or deacetylating enzymes by specific inhibitors. Recently, two microbial metabolites, trichostatin A and trapoxin, were found to be potent inhibitors of histone deacetylases. Trichostatin A reversibly inhibits the mammalian histone deacetylase, whereas trapoxin causes inhibition through irreversible binding to the enzyme. The histone deacetylase from a trichostatin A-resistant cell line is resistant to trichostatin A, indicating that the enzyme is the primary target. Both of the agents induce a variety of biological responses of cells such as induction of differentiation and cell cycle arrest. Trichostatin A and trapoxin are useful in analyzing the role of histone acetylation in chromatin structure and function as well as in determining the genes whose activities are regulated by histone acetylation.

Acetylation↗
PubMed1995

Secretion by Saccharomyces cerevisiae of rat apolipoprotein E as a fusion to Mucor rennin.

As the first step for production of rat apolipoprotein E (rApoE) in Saccharomyces cerevisiae, the rApoE cDNA was cloned and its nucleotide sequence was determined. When the intact rApoE gene including the presequence-encoding region was expressed under the control of the yeast GAL7 promoter, no protein immunoreactive with anti-rApoE antibody was detected either in the culture medium or inside the cells. For the purpose of the extracellular production of rApoE, three fusion genes were constructed in which the mature rApoE-encoding sequence was connected after the pre, prepro, and whole regions of the gene encoding a fungal aspartic proteinase, Mucor pusillus rennin (MPP), since MPP is efficiently secreted from recombinant S. cerevisiae containing the MPP gene. When these three fusion genes were expressed under the control of the GAL7 promoter, only one, encoding the mature rApoE connected to the whole MPP sequence, directed efficient secretion of the fused protein. The maximum yield of the fused protein secreted into the medium reached 11.8 mg/l and the calculated rApoE part was 5.3 mg in the fused protein. The excreted fusion protein was glycosylated at the original two sites in the MPP part. The fused protein was gradually degraded in the medium probably by proteases of the host cell, because no such degradation occurred in a yeast pep4mutant strain.

Amino Acid Sequence↗
PubMed1995

Autoregulators.

Explore the source record for details and available documents.

4-Butyrolactone↗
PubMed1995

Morphology of ras-transformed cells becomes apparently normal again with tyrosine kinase inhibitors without a decrease in the ras-GTP complex.

Radicicol, an inhibitor of protein-tyrosine kinase, was found to cause morphological reversion of v-Ha-ras-transformed NIH3T3 fibroblasts and T24 human urinary bladder carcinoma cells that contain an activated ras mutation. The network of actin stress fibers was restored during the treatment with radicicol. A similar morphological change was observed with another protein-tyrosine kinase inhibitor, herbimycin A. Radicicol did not cause any changes in the proportion of the active GTP binding form of p21ras or its subcellular localization. These results rule out the possibility that the morphological reversion by radicicol is due to direct or indirect inhibition of the p21ras function. Cycloheximide and actinomycin D inhibited the morphological change by radicicol, suggesting that the induced transcription of a gene(s) followed by de novo protein synthesis is required for suppression of the transformed phenotype in ras-transformed cells by tyrosine kinase inhibitors.

Animals↗
PubMed1995

Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6.

Pseudoazurin, a low molecular weight protein containing a single type I copper, functions as an electron donor to a copper-containing nitrite reductase (NIR) in a denitrifying bacterium Alcaligenes faecalis S-6. To elucidate the protein-protein interaction between these two copper-containing proteins, each of nine out of 13 lysine residues on the surface of pseudoazurin were independently replaced by alanine or aspartate, and the effects of the mutations on the interaction with NIR, as well as the physicochemical properties of pseudoazurin, were analyzed. All of the mutated pseudoazurins showed optical spectra and oxidation-reduction potentials almost identical to those of wild-type pseudoazurin, suggesting that none of the replacements of these lysine residues affected the environment around the type I copper site. Kinetic analysis of electron transfer between mutated pseudoazurins and NIR reveals that the lysine mutations have very little effect on the rate of electron transfer to NIR, but substitution at residues 10, 38, 57 and 77, all close to the copper site, substantially decreases the affinity of pseudoazurin for NIR. This suggests that pseudoazurin interacts with NIR through the region close to the type I copper site. The refined X-ray structures of Lys38Asp and Lys10Asp/Lys38Asp show that the molecular structure has indeed changed little. A new space group is observed for the Lys109Ala mutant crystal. Crystal packing interactions change for the Lys10Asp/Lys38Asp mutant but remain the same for Lys38Asp and Lys59Ala mutants.

Alanine↗
PubMed1995

Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH2-terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.

Acetobacter↗
PubMed1995

Cloning and characterization of the A-factor receptor gene from Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein control streptomycin production, streptomycin resistance, and aerial mycelium formation in Streptomyces griseus. The A-factor receptor protein (ArpA) was purified from a cell lysate of S. griseus IFO 13350. The NH2-terminal amino acid sequences of ArpA and lysyl endopeptidase-generated fragments were determined for the purpose of preparing oligonucleotide primers for cloning arpA by the PCR method. The arpA gene cloned in this way directed the synthesis of a protein having A-factor-specific binding activity when expressed in Escherichia coli under the control of the T7 promoter. The arpA gene was thus concluded to encode a 276-amino-acid protein with a calculated molecular mass of 29.1 kDa, as determined by nucleotide sequencing. The A-factor-binding activity was observed with a homodimer of ArpA. The NH2-terminal portion of ArpA contained an alpha-helix-turn-alpha-helix DNA-binding motif that showed great similarity to those of many DNA-binding proteins, which suggests that it exerts its regulatory function for the various phenotypes by directly binding to a certain key gene(s). Although a mutant strain deficient in both the ArpA protein and A-factor production overproduces streptomycin and forms aerial mycelium and spores earlier than the wild-type strain because of repressor-like behavior of ArpA, introduction of arpA into this mutant abolished simultaneously its streptomycin production and aerial mycelium formation. All of these data are consistent with the idea that ArpA acts as a repressor-type regulator for secondary metabolite formation and morphogenesis during the early growth phase and A-factor at a certain critical intracellular concentration releases the derepression, thus leading to the onset of secondary metabolism and aerial mycelium formation. The presence of ArpA-like proteins among Streptomyces spp., as revealed by PCR, together with the presence of A-factor-like compounds, suggests that a hormonal control similar to the A-factor system exists in many species of this genus.

4-Butyrolactone↗
PubMed1995

Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essentially required for aerial mycelium formation and streptomycin production in Streptomyces griseus. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this strain on a high-copy-number plasmid. Subcloning and nucleotide sequencing revealed that one open reading frame with 218 amino acids, named AmfC, served as a multicopy suppressor of the aerial mycelium-defective phenotype of the A-factor-deficient strain. The amfC gene did not restore A-factor or streptomycin production, indicating that amfC is involved in aerial mycelium formation independently of secondary metabolic function. Disruption of the chromosomal amfC gene in the wild-type S. griseus strain caused a severe reduction in the abundance of spores but no effect on the shape or size of the spores. The infrequent sporulation of the amfC disruptant was reversed by introduction of amfC on a plasmid. The amfC-defective phenotype was also restored by the orf1590 gene but not by the amfR-amfA-amfB gene cluster. Nucleotide sequences homologous to the amfC gene were distributed in all of 12 Streptomyces species tested, including Streptomyces coelicolor A3(2). The amfC homolog of S. coelicolor A3(2) was cloned and its nucleotide sequence was determined. The AmfC products of S. griseus and S. coelicolor A3(2) showed a 60% identity in their amino acid sequences. Introduction of the amfC gene of S. coelicolor A3(2) into strain HH1 induced aerial mycelium formation and sporulation, which suggests that both play the same functional role in morphogenesis in the strains.

4-Butyrolactone↗
PubMed1995

High level secretion by Saccharomyces cerevisiae of human apolipoprotein E as a fusion to Rhizomucor rennin.

As the first step for production of human apolipoprotein E (hApoE) in Saccharomyces cerevisiae, the hApoE cDNA was cloned in Escherichia coli, on the basis of the nucleotide sequence reported previously. When the hApoE cDNA including its pre-sequence-encoding region was expressed under the control of the GAL7 promoter, no protein immunoreactive with anti-hApoE antibody was detected either in the culture medium or inside the cells. For efficient production and secretion of hApoE in S. cerevisiae, the mature hApoE-encoding region was fused to the prepro-sequence region of Rhizomucor rennin (MPR) and to the whole MPR gene including its prepro- and mature-MPR regions. When the fusion gene consisting of the prepro-sequence-encoding region and hApoE regions was expressed in S. cerevisiae, no protein reactive with the anti-hApoE antibody was detected in any fraction of the yeast cells, probably due to rapid degradation of the hApoE protein by yeast proteases. On the other hand, when hApoE was expressed as a fusion to the whole MPR protein, a considerable amount of the fused protein was secreted into the medium. The prepro-sequence of MPR was correctly processed from the fused protein in the medium by autocatalytic activity of MPR and by a protease(s) of the host cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗