Biomedical subjects

Makoto Nishiyama

Publications and source records attributed to Makoto Nishiyama.

At least 19 recordsLinked to original sources

PubMed2006

Alteration of coenzyme specificity of lactate dehydrogenase from Thermus thermophilus by introducing the loop region of NADP(H)-dependent malate dehydrogenase.

Previously we found that replacement of seven amino acid residues in a loop region markedly shifted the coenzyme specificity of malate dehydrogenase from NAD(H) toward NADP(H). In the present study, we replaced the seven amino acid residues in the corresponding region of an NAD(H)-dependent lactate dehydrogenase with those of NADP(H)-dependent malate dehydrogenase, and examined the coenzyme specificity of the resulting mutant enzyme. Coenzyme specificity was significantly shifted by 399-fold toward NADPH when k cat/Km(coenzyme) was used as the measure of coenzyme specificity. The effect of the replacements on coenzyme specificity is discussed based on in silico simulation of the three-dimensional structure of the lactate dehydrogenase mutant.

Amino Acid Sequence↗

PubMed2006

Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant.

To elucidate the structural basis for the alteration of coenzyme specificity from NADH toward NADPH in a malate dehydrogenase mutant EX7 from Thermus flavus, we determined the crystal structures at 2.0 A resolution of EX7 complexed with NADPH and NADH, respectively. In the EX7-NADPH complex, Ser42 and Ser45 form hydrogen bonds with the 2'-phosphate group of the adenine ribose of NADPH, although the adenine moiety is not seen in the electron density map. In contrast, although Ser42 and Ser45 occupy a similar position in the EX7-NADH complex structure, both the adenine and adenine ribose moieties of NADH are missing in the map. These results and kinetic analysis of site-directed mutant enzymes indicate (1) that the preference of EX7 for NADPH over NADH is ascribed to the recognition of the 2'-phosphate group by two Ser and Arg44, and (2) that the adenine moiety of NADPH is not recognized in this mutant.

Bacterial Proteins↗

PubMed2006

Kinetics and product analysis of the reaction catalysed by recombinant homoaconitase from Thermus thermophilus.

HACN (homoaconitase) is a member of a family of [4Fe-4S] cluster-dependent enzymes that catalyse hydration/dehydration reactions. The best characterized example of this family is the ubiquitous ACN (aconitase), which catalyses the dehydration of citrate to cis-aconitate, and the subsequent hydration of cis-aconitate to isocitrate. HACN is an enzyme from the alpha-aminoadipate pathway of lysine biosynthesis, and has been identified in higher fungi and several archaea and one thermophilic species of bacteria, Thermus thermophilus. HACN catalyses the hydration of cis-homoaconitate to (2R,3S)-homoisocitrate, but the HACN-catalysed dehydration of (R)-homocitrate to cis-homoaconitate has not been observed in vitro. We have synthesized the substrates and putative substrates for this enzyme, and in the present study report the first steady-state kinetic data for recombinant HACN from T. thermophilus using a (2R,3S)-homoisocitrate dehydrogenase-coupled assay. We have also examined the products of the reaction using HPLC. We do not observe HACN-catalysed 'homocitrate dehydratase' activity; however, we have observed that ACN can catalyse the dehydration of (R)-homocitrate to cis-homoaconitate, but HACN is required for subsequent conversion of cis-homoaconitate into homoisocitrate. This suggests that the in vivo process for conversion of homocitrate into homoisocitrate requires two enzymes, in simile with the propionate utilization pathway from Escherichia coli. Surprisingly, HACN does not show any activity when cis-aconitate is substituted for the substrate, even though other enzymes from the alpha-aminoadipate pathway can accept analogous tricarboxylic acid-cycle substrates. The enzyme shows no apparent feedback inhibition by L-lysine.

Aconitate Hydratase↗

PubMed2006

FOG-1 represses GATA-1-dependent FcepsilonRI beta-chain transcription: transcriptional mechanism of mast-cell-specific gene expression in mice.

Cell-type-specific transcription of mouse high-affinity IgE receptor (FcepsilonRI) beta-chain is positively regulated by the transcription factor GATA-1. Although GATA-1 is expressed in erythroid cells, megakaryocytes, and mast cells, the expression of mouse FcepsilonRI beta-chain is restricted to mast cells. In the present study, we characterized the role of GATA-associated cofactor FOG-1 in the regulation of the FcepsilonRI beta-chain promoter. The expression levels of FOG-1, GATA-1, and beta-chain in each hematopoietic cell line were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. FOG-1 expression was higher in the beta-chain-negative hematopoietic progenitor cell line Ba/F3 than in the beta-chain-positive mast cell line PT18. By contrast, GATA-1 expression was similar when comparing the 2 cell lines. A transient reporter assay demonstrated that the beta-chain promoter functioned in PT18 but not in Ba/F3 and that the transcription activity of the beta-chain promoter in PT18 was markedly suppressed by overexpression of FOG-1. Although the activity of the beta-chain promoter, which was upregulated by coexpression of GATA-1, was significantly suppressed by coexpression of FOG-1 in the simian kidney CV-1 cells (beta-chain(-), GATA-1(-), and FOG-1(-)), the transactivation of the beta-chain promoter by the GATA-1 mutant V205G, which cannot bind FOG-1, was not affected by coexpression of FOG-1. Further, overexpression of FOG-1 in PT18 resulted in decreases in cell surface expression of FcepsilonRI and beta-chain transcription. Finally, suppression of FOG-1 expression using an siRNA approach resulted in increased beta-chain promoter activity in Ba/F3. These results suggest that FOG-1 expression level regulates the GATA-1-dependent FcepsilonRI beta-chain promoter.

Animals↗

PubMed2006

Involvement of the arginine repressor in lysine biosynthesis of Thermus thermophilus.

Lysine biosynthesis of Thermus thermophilus proceeds in a similar way to arginine biosynthesis, and some lysine biosynthetic enzymes from T. thermophilus so far investigated have the potential to function in arginine biosynthesis. These observations suggest that arginine might regulate the expression of genes for lysine biosynthesis. To test this hypothesis, the argR gene encoding the regulator of arginine biosynthesis was cloned from T. thermophilus and its function in lysine biosynthesis was analysed. The addition of arginine to the culture medium inhibited the growth of an arginase gene knockout mutant of T. thermophilus, which presumably accumulates arginine inside the cells. Arginine-dependent growth inhibition was not alleviated by the addition of ornithine, which is a biosynthetic intermediate of arginine and serves as a peptidoglycan component of the cell wall in T. thermophilus. However, the growth inhibition was cancelled either by the simultaneous addition of lysine and ornithine or by a knockout of the argR gene, suggesting the involvement of argR in regulation of lysine biosynthesis in T. thermophilus. Electrophoretic mobility shift assay and DNase I footprinting revealed that the ArgR protein specifically binds to the promoter region of the major lysine biosynthetic gene cluster. Furthermore, an alpha-galactosidase reporter assay for this promoter indicated that arginine repressed the promoter in an argR-dependent manner. These results indicate that lysine biosynthesis is regulated by arginine in T. thermophilus.

Arginase↗

PubMed2005

Crystal structure of NAD-dependent malate dehydrogenase complexed with NADP(H).

For better understanding of the coenzyme specificity in NAD-dependent MDH (tMDH) from Thermus flavus AT-62, we determined the crystal structures of tMDH-NADP(H) complex at maximally 1.65 A resolution. The overall structure is almost the same as that of the tMDH-NADH complex. However, NADP(H) binds to tMDH in the reverse orientation, where adenine occupies the position near the catalytic center and nicotinamide is positioned at the adenine binding site of the tMDH-NADH complex. Consistent with this, kinetic analysis of the malate-oxidizing reaction revealed that NADP(+) inhibited tMDH at high concentrations. This has provided the first evidence for the alternative binding mode of the nicotinamide coenzyme, that has pseudo-symmetry in its structure, in a single enzyme.

Binding Sites↗

PubMed2005

GATA-1 is required for expression of Fc{epsilon}RI on mast cells: analysis of mast cells derived from GATA-1 knockdown mouse bone marrow.

The high-affinity receptor for IgE (FcepsilonRI) that is expressed on the surface of mast cells plays an important role in antigen/IgE-mediated allergic reactions. We have previously found that critical elements in the promoter of the FcepsilonRI alpha- and beta-chain genes are recognized by the transcription factor GATA-1 in electrophoretic mobility shift assays coupled with a transient expression system for the alpha- and beta-chain promoters. To confirm that GATA-1 is involved in the expression of FcepsilonRI definitively, we generated bone marrow-derived mast cells from GATA-1 knockdown (KD) heterozygous mice. FACS analysis showed that the frequency of FcepsilonRI-positive cells was significantly decreased in mast cells derived from bone marrow of GATA-1 KD mice. Reverse transcription-PCR analysis showed that the level of transcripts not only for GATA-1 but also for both the alpha- and beta-chains was significantly lower in KD mast cells, whereas that of the FcepsilonRI gamma-chain was not affected. Degranulation caused by cross-linking of FcepsilonRI on mast cells prepared from KD mice was markedly repressed in comparison with that of wild-type mast cells. We concluded that the transcription factor GATA-1 positively regulates FcepsilonRI alpha- and beta-chain expression and therefore is involved in mast cell development.

Animals↗

PubMed2005

Leader peptide-mediated transcriptional attenuation of lysine biosynthetic gene cluster in Thermus thermophilus.

The molecular mechanism for regulation of the genes involved in the biosynthesis of amino acids is poorly identified in Thermus thermophilus. In this study, we analyzed the transcriptional control of the major lysine biosynthetic gene cluster in T. thermophilus. S1 nuclease mapping revealed that the transcription, which is repressed by lysine, starts at 111 bp, upstream of the translational start codon, ATG, for the homocitrate synthase (hcs) gene. The 5'-leader region of 111 bp carries a sequence that can encode a short peptide of 14 amino acids with tandem-arranged lysine residues in its sequence. The nucleotide sequence of the region suggests that the transcript can form complicated secondary structures. Deletion of most of the 5'-leader region or mutation of the tandem lysine codons suppressed the transcriptional repression by lysine. Mutation of the tandem codons from lysine to glutamine resulted in glutamine-dependent repression of the gene connected downstream, indicating that the leader peptide mediated the transcriptional attenuation of the gene expression. This is the first report demonstrating the transcriptional regulation of amino acid biosynthesis in T. thermophilus.

Bacillus subtilis↗

PubMed2005

Analysis of PU.1/ICSBP (IRF-8) complex formation with various PU.1 mutants: molecular cloning of rat Icsbp (Irf-8) cDNA.

We isolated cDNA encoding a full-length Interferon (IFN) consensus sequence binding protein [Icsbp; also called IFN regulatory factor-8 (Irf-8)] from rat and analyzed interaction between ICSBP and PU.1, an Ets-family transcription factor regulating hematopoietic cell-specific promoters. Electrophoretic mobility shift assay indicated that PU.1 with deletion of the PEST domain could not bind to ICSBP, although loss of the PEST domain had no effect on DNA-binding ability of PU.1 itself. An amino-acid replacement of Ser147 by Ala of the PEST domain of PU.1 did not affect DNA-binding ability of PU.1 to form a binary complex, PU.1/DNA, but clearly decreased the ternary complex formation of PU.1/ICSBP/DNA. Phosphatase treatment of the ternary complex markedly decreased PU.1/ICSBP/DNA-complex formation. These results indicated that Ser147 of PU.1 is definitively required for forming a complex with ICSBP and phosphorylation of PU.1 and/or ICSBP is critical for formation of the ternary complex.

Animals↗

PubMed2005

Mast cells acquire monocyte-specific gene expression and monocyte-like morphology by overproduction of PU.1.

PU.1 is a myeloid- and lymphoid-specific transcription factor that belongs to the Ets family. Recently, we found that overproduction of PU.1 in mouse bone marrow-derived hemopoietic progenitor cells induced monocyte-specific gene expression and caused their monocyte-like morphological change. In the present study, PU.1 was overproduced by using retrovirus expression system in differentiated bone marrow-derived mast cells. By overexpression of PU.1, cell surface expression of MHC class II, CD11b, CD11c, and F4/80 was induced, accompanied by reduced expression of c-kit, a mast cell-specific marker. Morphology of PU.1-transfected cells was altered toward monocyte-like one. PU.1-overproducing cells acquired T cell stimulatory ability and showed an increase in response to LPS stimulation, while response through FcepsilonRI was markedly reduced by overproduction of PU.1. These results suggest that the differentiated mast cells still have potential to display monocytic features. When PU.1 was overproduced in a different type of mast cell, peritoneal mast cells, similar monocyte-like morphological change, and the expression of CD11b and F4/80 were induced. However, surface level of CD11c and MHC class II was not affected. These results indicate that the potential capacity to exhibit monocytic features is different between both the mast cells.

Animals↗

PubMed2005

Crystal structure of tetrameric homoisocitrate dehydrogenase from an extreme thermophile, Thermus thermophilus: involvement of hydrophobic dimer-dimer interaction in extremely high thermotolerance.

The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-A resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related beta-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.

Alcohol Oxidoreductases↗

PubMed2004

RERJ1, a jasmonic acid-responsive gene from rice, encodes a basic helix-loop-helix protein.

Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with jasmonic acid (JA) for 1/2h yielded a cDNA of a gene tentatively named RERJ1 that is upregulated in response to exogenous JA. Northern blot analysis indicated that the RERJ1 mRNA levels peaked at 1/2-1h after the addition of jasmonic acid and then decreased gradually. RERJ1 encodes a transcriptional regulator with a basic helix-loop-helix motif. The phenotypes of transgenic rice plants overexpressing sense or antisense RERJ1 mRNA demonstrated that RERJ1 is involved in the growth inhibition of rice shoots caused by JA. Other biological functions of RERJ1 are discussed from an evolutionary standpoint.

Amino Acid Sequence↗

PubMed2004

Polymorphisms in the Fc epsilon RI beta promoter region affecting transcription activity: a possible promoter-dependent mechanism for association between Fc epsilon RI beta and atopy.

The beta subunit of the high-affinity IgE receptor (FcepsilonRI) plays an important role in IgE-mediated allergic reactions as an amplifier for cell surface expression and signal transduction of FcepsilonRI. FcepsilonRIbeta is presumed to be one of the genes linked with atopic diseases. However, the validity of the associations previously found between single nucleotide polymorphisms (SNPs) in FcepsilonRIbeta and atopic diseases is questionable. In the present study, we found correlation between the SNP of FcepsilonRIbeta at +6960A/G, resulting in a Glu237Gly amino acid substitution, and the cell surface expression level of FcepsilonRI on blood basophils, although it has been shown that the Glu237Gly mutation itself does not affect the surface expression or function of FcepsilonRI. We additionally found four SNPs in the promoter region of FcepsilonRIbeta, among which -426T/C and -654C/T were tightly linked with +6960A/G. Reporter plasmids carrying the -426C and -654T promoter displayed higher transcriptional activity than those carrying the -426T and -654C promoter. We found that transcription factor YY1 preferentially bound and transactivated the -654T promoter. Furthermore, expression of FcepsilonRI beta-chain mRNA in basophils from individuals who have the minor heterozygous genotype was significantly higher than that of the major homozygous genotype. These results suggest that the SNPs in the FcepsilonRIbeta promoter are causally linked with atopy via regulation of FcepsilonRI expression.

5' Flanking Region↗

PubMed2004

Evidence against requirement of Ser41 and Ser45 for function of PU.1 -- molecular cloning of rat PU.1.

The transcription factor PU.1 plays an important role in the development of the myeloid and lymphoid lineages and regulates the transcription of several genes expressed in these cells. Ser41 is conserved in the acidic region (33-47) of PU.1 from a variety of eukaryocytes and has been reported to be one of the two important Ser residues (S41 and S45) for the function of PU.1. In the present study, however, we found that rat PU.1 has Gly at position 41. To elucidate the role of amino acid residues at 41 and 45 in functions of PU.1, we generated mutants of rat PU.1, G41S, G41A, and S45A, and analyzed their transcription-enhancing activities by using two different systems, transient reporter assay system and retroviral transfection system. The amino acid substitution at 41 of PU.1 causes no effect on both transcription-enhancing activity for M-CSF receptor promoter and the cooperative transcription-enhancing activity with GATA-1 for FcRI alpha-chain promoter. Furthermore, the substitution at 41 also had no effect on the activity to induce monocyte-specific gene expression in the bone marrow-derived hematopoietic cells. From these results, we conclude that Ser41 as well as Ser45 are not essential for the promoter-upregulating function of PU.1.

Amino Acid Sequence↗

PubMed2004

Stemar-13-ene synthase, a diterpene cyclase involved in the biosynthesis of the phytoalexin oryzalexin S in rice.

In suspension-cultured rice cells, diterpenoid phytoalexins are produced in response to exogenously applied elicitors. We isolated a cDNA encoding a diterpene cyclase, OsDTC2, from suspension-cultured rice cells treated with a chitin elicitor. The OsDTC2 cDNA was overexpressed in Escherichia coli as a fusion protein with glutathione S-transferase, and the recombinant OsDTC2 was indicated to function as stemar-13-ene synthase that converted syn-copalyl diphosphate to stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S. The level of OsDTC2 mRNA in suspension-cultured rice cells began to increase 3 h after addition of the elicitor and reached the maximum after 8 h. The expression of OsDTC2 was also induced in UV-irradiated rice leaves. In addition, we indicated that stemar-13-ene accumulated in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves.

Base Sequence↗

PubMed2004

Functional analysis of the small subunit of the putative homoaconitase from Pyrococcus horikoshii in the Thermus lysine biosynthetic pathway.

An in vivo disruption-integration vector system for Thermus thermophilus was developed and used for the functional analysis of an evolutionary-related archaeal protein for lysine biosynthesis. In contrast to fungal one, the putative homoaconitase of T. thermophilus consists of two subunits and catalyzes the second and third steps of lysine biosynthesis. ORFs from hyperthermophilic archaeon Pyrococcus horikoshii, PH1726 and PH1724, share a high degree of amino acid identity with the T. thermophilus subunits LysT and LysU, respectively. In the present report, gene encoding the putative small subunit of archaeal homoaconitase, PH1724, was integrated into the lysU locus of T. thermophilus. The archaeal gene was expressed under the control of PslpA promoter and functional analyses were performed. Transformants were able to grow on minimal medium without lysine when PH1724 ORF was integrated, whereas the lysU disruption led to lysine auxotrophy. Chromosomal integration was verified by PCR analysis, and homoaconitase assay showed that the archaeal gene product functions as a small subunit of homoaconitase, possibly by forming a heterodimer with the LysT subunit of T. thermophilus. These results strongly suggest the functional relation of P. horikoshii PH1724 with LysU in the Thermus lysine biosynthetic pathway, together with functional assignment of LysU as small subunit of homoaconitase. In addition, the provided results indicate that archaeal genes products from hyperthermophiles can be studied in a thermophilic eubacterium such as T. thermophilus.

Amino Acid Sequence↗

PubMed2004

Conversion of feedback regulation in aspartate kinase by domain exchange.

To elucidate the mechanism for the regulation of aspartate kinase (AK) via feedback inhibition, we constructed several chimeric enzymes between Bacillus subtilis AK II, a lysine-sensitive mesophilic enzyme, and Thermus flavus AK, a threonine-sensitive thermostable enzyme, each having the same alpha2beta2-type tetrameric structure. A chimeric AK, named BTT, composed of the chimeric alpha subunit that comprises of the N-terminal catalytic region from B. subtilis AK II and the C-terminal region from T. flavus, and the beta subunit from T. flavus, was inhibited only by threonine. Another chimeric enzyme, BT, which has a similar structure to that of BTT but lacks the beta subunit, having alpha2-type homo-dimeric structure, was also responsive only to threonine. However, the addition of threonine enhanced the activity of BT. These results indicate the regulatory function of C-terminal region and beta subunit in AK. BTT showed extremely high thermostability comparable to that of T. flavus, suggesting that the beta subunit also contributed to the stability of the AK.

Amino Acid Sequence↗

PubMed2004

Functional analysis of PU.1 domains in monocyte-specific gene regulation.

The Ets family transcription factor PU.1 is required for the development of various lymphoid and myeloid cell lineages, and regulates the expression of several genes in a cell type-specific manner. Recently we found that overproduction of PU.1 in mouse bone marrow-derived mast cell progenitors induced the expression of monocyte-specific genes. This prompted us to analyze the functions of each domain of PU.1 in monocyte-specific gene expression, using transfection of mast cell progenitors with a series of retrovirus vectors for overexpression of various truncation mutants. Both the acidic region and the Ets domain of PU.1 were required for expression of monocyte-specific genes, and for enhanced interleukin-6 production in response to lipopolysaccharide. The Gln-rich region was suggested to be involved in expression of both MHC class II and F4/80. On the other hand, when PU.1 protein lacking the PEST domain was produced in the progenitor cells, expression of monocyte-specific genes was substantially enhanced, suggesting that the PEST domain plays a negative role in monocyte-specific gene expression.

Animals↗