Biomedical subjects

Sueharu Horinouchi

Publications and source records attributed to Sueharu Horinouchi.

At least 19 recordsLinked to original sources

PubMed2006

Multiple histone deacetylases and the CREB-binding protein regulate pre-mRNA 3'-end processing.

Trichostatin A (TSA), a specific inhibitor of histone deacetylases (HDACs), induces acetylation of various non-histone proteins such as p53 and alpha-tubulin. We purified several acetylated proteins by the affinity to an anti-acetylated lysine (AcLys) antibody from cells treated with TSA and identified them by mass spectrometry. Here we report on acetylation of CFIm25, a component of mammalian cleavage factor Im (CF Im), and poly(A) polymerase (PAP), a polyadenylating enzyme for the pre-mRNA 3'-end. The residues acetylated in these proteins were mapped onto the regions required for interaction with each other. Whereas CBP acetylated these proteins, HDAC1, HDAC3, HDAC10, SIRT1, and SIRT2 were involved in in vivo deacetylation. Acetylation of the CFIm25 occurred depending on the cleavage factor complex formation. Importantly, the interaction between PAP and CF Im complex was decreased by acetylation. We also demonstrated that acetylation of PAP inhibited the nuclear localization of PAP by inhibiting the binding to the importin alpha/beta complex. These results suggest that CBP and HDACs regulate the 3'-end processing machinery and modulate the localization of PAP through the acetylation and deacetylation cycle.

3' Untranslated Regions↗

PubMed2006

Arylamine N-acetyltransferase responsible for acetylation of 2-aminophenols in Streptomyces griseus.

An arylamine N-acetyltransferase (NAT) responsible for the N acetylation of exogenous 3-amino-4-hydroxybenzoic acid in Streptomyces griseus was identified and characterized. This enzyme was distinct from other eukaryotic and bacterial NATs in that it acetylated various 2-aminophenol derivatives more effectively than it acetylated 5-aminosalicylic acid, and thus it may be involved in the metabolism of xenobiotic compounds.

Acetylation↗

PubMed2006

Novel benzene ring biosynthesis from C(3) and C(4) primary metabolites by two enzymes.

The shikimate pathway, including seven enzymatic steps for production of chorismate via shikimate from phosphoenolpyruvate and erythrose-4-phosphate, is common in various organisms for the biosynthesis of not only aromatic amino acids but also most biogenic benzene derivatives. 3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a benzene derivative serving as a precursor for several secondary metabolites produced by Streptomyces, including grixazone produced by Streptomyces griseus. Our study on the biosynthesis pathway of grixazone led to identification of the biosynthesis pathway of 3,4-AHBA from two primary metabolites. Two genes, griI and griH, within the grixazone biosynthesis gene cluster were found to be responsible for the biosynthesis of 3,4-AHBA; the two genes conferred the in vivo production of 3,4-AHBA even on Escherichia coli. In vitro analysis showed that GriI catalyzed aldol condensation between two primary metabolites, l-aspartate-4-semialdehyde and dihydroxyacetone phosphate, to form a 7-carbon product, 2-amino-4,5-dihydroxy-6-one-heptanoic acid-7-phosphate, which was subsequently converted to 3,4-AHBA by GriH. The latter reaction required Mn(2+) ion but not any cofactors involved in reduction or oxidation. This pathway is independent of the shikimate pathway, representing a novel, simple enzyme system responsible for the synthesis of a benzene ring from the C(3) and C(4) primary metabolites.

Aminobenzoates↗

PubMed2006

One-pot synthesis of genistein from tyrosine by coincubation of genetically engineered Escherichia coli and Saccharomyces cerevisiae cells.

For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as "enzyme bags" and incubated at 30 degrees C for 48 h in 100 ml of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 microg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium glutamicum, all of which were under the control of the isopropyl-beta-D-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S. cerevisiae cell containing the IFS gene. Coincubation of the E. coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26 degrees C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate yielded ca. 6 mg genistein/l.

Acetyl-CoA Carboxylase↗

PubMed2006

ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe.

Cloning of the entire set of an organism's protein-coding open reading frames (ORFs), or 'ORFeome', is a means of connecting the genome to downstream 'omics' applications. Here we report a proteome-scale study of the fission yeast Schizosaccharomyces pombe based on cloning of the ORFeome. Taking advantage of a recombination-based cloning system, we obtained 4,910 ORFs in a form that is readily usable in various analyses. First, we evaluated ORF prediction in the fission yeast genome project by expressing each ORF tagged at the 3' terminus. Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. Furthermore, using leptomycin B, an inhibitor of the nuclear export protein Crm1, we identified 285 proteins whose localization is regulated by Crm1.

Antifungal Agents↗

PubMed2006

Phenolic lipid synthesis by type III polyketide synthases is essential for cyst formation in Azotobacter vinelandii.

Cysts of Azotobacter vinelandii are resting cells that are surrounded by a protective coat, conferring resistance to various chemical and physical agents. The major chemical components of the cyst coat are alkylresorcinols, which are amphiphilic molecules possessing an aromatic ring with a long aliphatic carbon chain. Although alkylresorcinols are widely distributed in bacteria, fungi, plants, and animals, no enzyme systems for their biosynthesis are known. We report here an ars operon in A. vinelandii that is responsible for the biosynthesis of the alkylresorcinols in the cysts. The ars operon consisted of four genes, two of which encoded a type III polyketide synthase, ArsB and ArsC. In vitro experiments revealed that ArsB and ArsC, sharing 71% amino acid sequence identity, were an alkylresorcinol synthase and an alkylpyrone synthase, respectively, indicating that ArsB and ArsC are not isozymes but enzymatically distinct polyketide synthases. In addition, ArsB and ArsC accepted several acyl-CoAs with various lengths of the side chain as a starter substrate and gave corresponding alkylresorcinols and alkylpyrones, respectively, which suggests that the mode of the ring folding is uninfluenced by the structure of the starter substrates. The importance of the alkylresorcinols for encystment was confirmed by gene inactivation experiments; the lack of alkylresorcinols synthesis caused by ars mutations resulted in the formation of severely impaired cysts, as observed by electron microscopy.

Azotobacter vinelandii↗

PubMed2006

Self-activation of serine/threonine kinase AfsK on autophosphorylation at threonine-168.

A Hanks-type protein kinase AfsK autophosphorylates on threonine residue(s) and phosphorylates AfsR, a global regulator for secondary metabolism in Streptomyces coelicolor A3(2). Mass spectrometry of a tryptic digest of the autophosphorylated form of AfsK deltaC corresponding to the kinase catalytic domain (Met-1 to Arg-311) of AfsK, together with subsequent site-directed mutagenesis of the candidate amino acids, identified threonine-168 as a single autophosphorylation site. Threonine-168 is located in the activation loop that is known for some Ser/Thr kinases to modulate kinase activity on phosphorylation of one or more threonine residues within the loop. Consistent with this, mutant T168D, in which Thr-168 was replaced by Asp, became a constitutively active kinase; it phosphorylated AfsR to the same extent as AfsK deltaC produced in and purified from Escherichia coli cells during which a considerable population of it had been already phosphorylated intermolecularly. All these findings show that autophosphorylation or intermolecular phosphorylation of threonine-168 in AfsK accounts for the self-activation of its kinase activity.

Amino Acid Sequence↗

PubMed2006

Chitinase inhibitor allosamidin is a signal molecule for chitinase production in its producing Streptomyces II. Mechanism for regulation of chitinase production by allosamidin through a two-component regulatory system.

In Streptomyces sp. AJ9463, a producer of chitinase inhibitor allosamidin, allosamidin strongly enhances production of the chitinase mainly secreted to the culture broth in a chitin medium. To clarify the mechanism for regulation of the chitinase production by allosamidin, a disruption experiment of genes encoding proteins constructing a two-component regulatory system present at 5'-upstream region of the chitinase gene was performed. In the disruptant obtained, allosamidin could not promote the chitinase production, but N, N'-diacetylchitobiose, which also enhances production of the same chitinase more weakly than allosamidin, promoted the chitinase production similarly to the case observed in the wild-type strain. Furthermore, by the experiment in an inorganic salt solution, it was shown that allosamidin could not induce the chitinase production without addition of N, N'-diacetylchitobiose. These results show that allosamidin can activate transcription of the chitinase gene through the two-component regulatory system in the presence of N, N'-diacetylchitobiose.

Acetylglucosamine↗

PubMed2006

Putative ABC transporter responsible for acetic acid resistance in Acetobacter aceti.

Two-dimensional gel electrophoretic analysis of the membrane fraction of Acetobacter aceti revealed the presence of several proteins that were produced in response to acetic acid. A 60-kDa protein, named AatA, which was mostly induced by acetic acid, was prepared; aatA was cloned on the basis of its NH2-terminal amino acid sequence. AatA, consisting of 591 amino acids and containing ATP-binding cassette (ABC) sequences and ABC signature sequences, belonged to the ABC transporter superfamily. The aatA mutation with an insertion of the neomycin resistance gene within the aatA coding region showed reduced resistance to acetic acid, formic acid, propionic acid, and lactic acid, whereas the aatA mutation exerted no effects on resistance to various drugs, growth at low pH (adjusted with HCl), assimilation of acetic acid, or resistance to citric acid. Introduction of plasmid pABC101 containing aatA under the control of the Escherichia coli lac promoter into the aatA mutant restored the defect in acetic acid resistance. In addition, pABC101 conferred acetic acid resistance on E. coli. These findings showed that AatA was a putative ABC transporter conferring acetic acid resistance on the host cell. Southern blot analysis and subsequent nucleotide sequencing predicted the presence of aatA orthologues in a variety of acetic acid bacteria belonging to the genera Acetobacter and Gluconacetobacter. The fermentation with A. aceti containing aatA on a multicopy plasmid resulted in an increase in the final yield of acetic acid.

ATP-Binding Cassette Transporters↗

PubMed2006

Control of the Streptomyces Subtilisin inhibitor gene by AdpA in the A-factor regulatory cascade in Streptomyces griseus.

AdpA in the A-factor regulatory cascade in Streptomyces griseus activates a number of genes required for secondary metabolism and morphological differentiation, forming an AdpA regulon. The Streptomyces subtilisin inhibitor (SSI) gene, sgiA, in S. griseus was transcribed in response to AdpA, showing that sgiA is a member of the AdpA regulon. AdpA bound a single site upstream of the sgiA promoter at approximately position -70 with respect to its transcriptional start point. Mutational analysis of the AdpA-binding site showed that the AdpA-binding site was essential for transcriptional activation. Mutants in which sgiA was disrupted had higher trypsin, chymotrypsin, metalloendopeptidase, and total protease activities than the wild-type strain, which showed that SgiA modulated the activities of these extracellularly produced proteases. Because a number of genes encoding chymotrypsins, trypsins, and metalloendopeptidases, most of which are SSI-sensitive proteases, are also under the control of AdpA, the A-factor regulatory cascade was thought to play a crucial role in modulating the extracellular protease activities by triggering simultaneous production of the proteases and their inhibitor at a specific timing during growth. Mutants in which sgiA was disrupted grew normally and formed aerial hyphae and spores with the same time course as the wild-type strain. However, exogenous addition of purified SgiA to substrate mycelium grown on agar medium resulted in a delay in aerial mycelium formation, indicating that SgiA is involved in aerial hypha formation in conjunction with proteases.

Amino Acid Sequence↗

PubMed2006

Induction of a ribotoxic stress response that stimulates stress-activated protein kinases by 13-deoxytedanolide, an antitumor marine macrolide.

13-Deoxytedanolide is a structurally unique macrolide with strong antitumor activity isolated from a marine sponge. Recently, we showed that 13-deoxytedanolide bound to the large subunit of the yeast ribosome and inhibited polypeptide elongation in vitro, but the mechanism by which it exerts antitumor activity is still unknown. Here we show that 13-deoxytedanolide strongly induces plasminogen activator inhibitor 1 (PAI-1) promoter-derived gene expression. 13-Deoxytedanolide, unlike TGF-beta, did not cause apparent nuclear translocation of Smad2/3, but it relocalized the temperature-sensitive mutant of mouse p53 (p53Val153) from the cytoplasm to the nucleus at a nonpermissive temperature, suggesting that 13-deoxytedanolide inhibits protein synthesis. Indeed, the drug inhibited in vivo protein synthesis at low nanomolar concentrations and strongly activated stress-activated protein kinases such as p38 mitogen-activated protein kinase and Jun NH2-terminal protein kinase (JNK). Anisomycin, a well-known inducer of ribotoxic stress that activates both p38 and JNK, also activated PAI-1 gene expression, while other protein synthesis inhibitors that do not activate the kinases failed to do so. PAI-1 gene expression by 13-deoxytedanolide and anisomycin was blocked by SB202190, a specific inhibitor of p38, and SP600125, an inhibitor of both p38 and JNK. 13-Deoxytedanolide and anisomycin caused activation of apoptosis signal-regulating kinase 1, MKK3/MKK6, and SEK1/MKK4, the regulatory kinases upstream of p38 and JNK. These results suggest that 13-deoxytedanolide, like anisomycin, triggers a ribotoxic stress response that activates stress-activated protein kinase cascades, thereby inducing PAI-1 gene expression and apoptosis.

Animals↗

PubMed2005

A novel o-aminophenol oxidase responsible for formation of the phenoxazinone chromophore of grixazone.

Grixazone contains a phenoxazinone chromophore and is a secondary metabolite produced by Streptomyces griseus. In the grixazone biosynthesis gene cluster, griF (encoding a tyrosinase homolog) and griE (encoding a protein similar to copper chaperons for tyrosinases) are encoded. An expression study of GriE and GriF in Escherichia coli showed that GriE activated GriF by transferring copper ions to GriF, as has been observed for a Streptomyces melanogenesis system in which the MelC1 copper chaperon transfers copper ions to MelC2 tyrosinase. In contrast with tyrosinases, GriF showed no monophenolase activity, although it oxidized various o-aminophenols as preferable substrates rather than catechol-type substrates. Deletion of the griEF locus on the chromosome resulted in accumulation of 3-amino-4-hydroxybenzaldehyde (3,4-AHBAL) and its acetylated compound, 3-acetylamino-4-hydroxybenzaldehyde. GriF oxidized 3,4-AHBAL to yield an o-quinone imine derivative, which was then non-enzymatically coupled with another molecule of the o-quinone imine to form a phenoxazinone. The coexistence of N-acetylcysteine in the in vitro oxidation of 3,4-AH-BAL by GriF resulted in the formation of grixazone A, suggesting that the -SH group of N-acetylcysteine is conjugated to the o-quinone imine formed from 3,4-AHBAL and that the conjugate is presumably coupled with another molecule of the o-quinone imine. GriF is thus a novel o-aminophenol oxidase that is responsible for the formation of the phenoxazinone chromophore in the grixazone biosynthetic pathway.

Acetylcysteine↗

PubMed2005

Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster.

For the fermentative production of plant-specific flavanones (naringenin, pinocembrin) by Escherichia coli, a plasmid was constructed which carried an artificial biosynthetic gene cluster, including PAL encoding a phenylalanine ammonia-lyase from a yeast, ScCCL encoding a cinnamate/coumarate:CoA ligase from the actinomycete Streptomyces coelicolor A3(2), CHS encoding a chalcone synthase from a licorice plant and CHI encoding a chalcone isomerase from the Pueraria plant. The recombinant E. coli cells produced (2S)-naringenin from tyrosine and (2S)-pinocembrin from phenylalanine. When the two subunit genes of acetyl-CoA carboxylase from Corynebacterium glutamicum were expressed under the control of the T7 promoter and the ribosome-binding sequence in the recombinant E. coli cells, the flavanone yields were greatly increased, probably because enhanced expression of acetyl-CoA carboxylase increased a pool of malonyl-CoA that was available for flavanone synthesis. Under cultural conditions where E. coli at a cell density of 50 g/l was incubated in the presence of 3 mM tyrosine or phenylalanine, the yields of naringenin and pinocembrin reached about 60 mg/l. The fermentative production of flavanones in E. coli is the first step in the construction of a library of flavonoid compounds and un-natural flavonoids in bacteria.

Escherichia coli↗

PubMed2005

Combinatorial biosynthesis of flavones and flavonols in Escherichia coli.

(2S)-Flavanones (naringenin and pinocembrin) are key intermediates in the flavonoid biosynthetic pathway in plants. Recombinant Escherichia coli cells containing four genes for a phenylalanine ammonia-lyase, cinnamate/coumarate:CoA ligase, chalcone synthase, and chalcone isomerase, in addition to the acetyl-CoA carboxylase, have been established for efficient production of (2S)-naringenin from tyrosine and (2S)-pinocembrin from phenylalanine. Further introduction of the flavone synthase I gene from Petroselinum crispum under the control of the T7 promoter and the synthetic ribosome-binding sequence in pACYCDuet-1 caused the E. coli cells to produce flavones: apigenin (13 mg/l) from tyrosine and chrysin (9.4 mg/l) from phenylalanine. Introduction into the E. coli cells of the flavanone 3beta-hydroxylase and flavonol synthase genes from the plant Citrus species led to production of flavonols: kaempferol (15.1 mg/l) from tyrosine and galangin (1.1 mg/l) from phenylalanine. The combinatorial biosynthesis of the flavones and flavonols in E. coli is promising for the construction of a library of various flavonoid compounds and un-natural flavonoids in bacteria.

Citrus↗

PubMed2005

Autorepression of AdpA of the AraC/XylS family, a key transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus.

AdpA belonging to the AraC/XylS family is a key transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus, activating a number of genes required for physiological and morphological differentiation. On the other hand, AdpA repressed its own transcription by cooperative binding to the promoter region containing multiple operator sites. AdpA contained three operator sites, site 1 approximately at nucleotide position -100, site 2 at the promoter elements, and site 3 at position +80. AdpA bound to a strong binding site 1 increased the affinity for AdpA of a weak site 2, probably by forming a DNA loop via the two molecules of AdpA dimer, thus preventing RNA polymerase from access to the promoter. AdpA bound to site 3 with rather weak affinity repressed the AdpA promoter activity independently of sites 1 and 2, perhaps preventing RNA polymerase from chain elongation. Consistent with this model, the in vivo transcription of AdpA containing mutated site 1 or site 3 was greatly increased. In addition, streptomycin production, one of the phenotypes controlled positively by AdpA, was greatly increased in the mutants containing AdpA with a mutation at site 1 and site 3. The in vitro transcription of AdpA containing mutated site 1 was also increased. Thus, the transcription of AdpA, encoding an important transcriptional factor for ordered physiological and morphological development, is self-controlled.

Bacterial Proteins↗

PubMed2005

A calmodulin-like protein in the bacterial genus Streptomyces.

The gene, named cabB, encoding a calmodulin-like protein of 70 amino acids containing two helix-loop-helix EF-hand motifs was cloned from Streptomyces coelicolor A3(2). cabB was transcribed from a single promoter throughout growth. The CabB protein produced in Escherichia coli was a monomer in solution, although it corresponded to one half of a dumbbell shape of the eukaryotic calmodulins. CabB bound calcium and upon binding of calcium its alpha-helix content was increased, as determined by circular dichroism spectroscopy. The growth of cabB-disruptants (mutant DeltacabB) on minimal agar medium containing calcium higher than 20 mM was delayed, suggesting that CabB has a role in calcium homeostasis by serving as a calcium buffer or transporter, as suggested for calerythrin in actinomycetes and the invertebrate sarcoplasmic calcium-binding proteins. Wide distribution of cabB almost exclusively in actinomycetes suggests a common role of EF-hand CabB-type proteins in these filamentous, soil-dwelling Gram-positive bacterial genera.

Calcium↗

PubMed2005

A novel quinone-forming monooxygenase family involved in modification of aromatic polyketides.

RppA is a type III polyketide synthase (PKS) that catalyzes condensation of five molecules of malonyl-CoA to form 1,3,6,8-tetrahydroxynaphthalene (THN). In Streptomyces antibioticus IFO13271 and several other Streptomyces species, an open reading frame, named momA, is present as a neighbor of rppA. MomA belonged to the "cupin" superfamily because it contained a set of two motifs that is responsible for binding one equivalent of metal ions. MomA catalyzed monooxygenation of the THN produced from malonyl-CoA by the action of RppA to form flaviolin. In addition, it used several polyketides as substrates and formed the corresponding quinones. MomA required redox-active transition metal ions (Ni(2+), Cu(2+), Fe(3+), Fe(2+), Mn(2+), and Co(2+)) for its activity, whereas it was inhibited by a redox-inert transition metal ion (Zn(2+)). MomA neither possessed any flavin prosthetic group nor required nicotinamide cofactors for monooxygenation, which shows that MomA as a member of the cupin superfamily is a novel monooxygenase. Consistent with the catalytic property of MomA, WhiE-ORFII showing similarity in amino acid sequence to MomA and containing a cupin domain also catalyzed monooxygenation of THN. whiE-ORFII is located immediately upstream of the "minimal PKS" gene within the whiE type II PKS gene cluster for biosynthesis of a gray spore pigment in Streptomyces coelicolor A3(2), and a number of whiE-ORFII homologues are present in the biosynthetic gene cluster for polyketides of type II in various Streptomyces species. These findings show that a novel class of quinone-forming monooxygenases is involved in modification of aromatic polyketides synthesized by PKSs of types II and III.

Amino Acid Sequence↗

PubMed2005

Inhibition of protein synthesis and activation of stress-activated protein kinases by onnamide A and theopederin B, antitumor marine natural products.

During the course of screening for the agents that activate transforming growth factor-beta (TGF-beta) signaling cascade, onnamide A and theopederin B, heterocyclic compounds related to mycalamides from a marine sponge, were found to induce plasminogen activator inhibitor-1 (PAI-1) promoter-derived gene expression in Mv1Lu cells. The maximum induction of the PAI-1 promoter by onnamide A and theopederin B was observed at the concentrations of 50 nM and 2 nM, respectively. These compounds strongly inhibited protein synthesis at the 50% inhibitory concentrations of 30 nM for onnamide A and 1.9 nM for theopederin B, and induced activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal protein kinase (JNK). Anisomycin, a well-known inducer of ribotoxic stress that inhibits protein synthesis and activates both p38 kinase and JNK, also activated PAI-1 gene expression. Furthermore, PAI-1 expression by onnamide A, theopederin B, and anisomycin was inhibited by SB202190 and SP600125, specific inhibitors of stress-activated protein kinases. Onnamide A and theopederin B were cytotoxic to a variety of cell lines and strongly induced apoptosis in HeLa cells within 24 h, which was accompanied by the sustained activation of p38 kinase and JNK. These results suggest that onnamide A and theopedirin B trigger a ribotoxic stress-like response, thereby inducing p38 kinase and JNK activation, the kinase-dependent PAI-1 gene expression, and apoptosis.

Animals↗