Tomohisa Kuzuyama

The University of Tokyo, Edo, Tōkyō, Japan

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Publications (124)459.51 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: ArgR is known to serve as a repressor/activator of the metabolism of arginine. To elucidate the role of ArgR in the metabolism of Thermus thermophilus cells, comparative genome-wide comprehensive analysis was conducted for wild-type T. thermophilus and its mutant lacking the argR gene. Transcriptome analysis and chromatin affinity precipitation coupled with high-density tiling chip (ChAP-chip) analysis identified 34 genetic loci that are directly regulated by ArgR and indicated that ArgR decreases the expression of arginine biosynthesis and also regulates several other genes involved in amino acid metabolism, including lysine biosynthetic genes, as suggested by our previous study. Among genes whose expression was regulated by ArgR, the largest effect of argR knockout was observed in a putative operon, including genes TTHA0284, TTHA0283, and TTHA0282 involved in arginine biosynthesis. The promoter of this operon, argG, was repressed approximately 21-fold by ArgR. DNase I footprint analysis coupled with electrophoretic mobility shift assay suggested that high arginine-dependent repression was attributed to the fact that the promoter contains three operators for ArgR binding and ArgR is bound to the binding sites cooperatively, possibly forming a DNA loop, in the hexameric form stabilized by arginine binding.
    Extremophiles : life under extreme conditions. 07/2014;
  • The Journal of antibiotics. 07/2014;
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    ABSTRACT: Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing foreign (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a metabolite in the 2-C-methyl-d-erythritol-4-phosphate pathway used by most eubacteria and apicomplexan parasites, and self isopentenyl pyrophosphate, a metabolite in the mevalonate pathway used by humans. Whereas microbial infections elicit prolonged expansion of memory Vγ2Vδ2 T cells, immunization with prenyl pyrophosphates or aminobisphosphonates elicit short-term Vγ2Vδ2 expansion with rapid anergy and deletion upon subsequent immunizations. We hypothesized that a live, attenuated bacterial vaccine that overproduces HMBPP would elicit long-lasting Vγ2Vδ2 T cell immunity by mimicking a natural infection. Therefore, we metabolically engineered the avirulent aroA(-) Salmonella enterica serovar Typhimurium SL7207 strain by deleting the gene for LytB (the downstream enzyme from HMBPP) and functionally complementing for this loss with genes encoding mevalonate pathway enzymes. LytB(-) Salmonella SL7207 had high HMBPP levels, infected human cells as efficiently as did the wild-type bacteria, and stimulated large ex vivo expansions of Vγ2Vδ2 T cells from human donors. Importantly, vaccination of a rhesus monkey with live lytB(-) Salmonella SL7207 stimulated a prolonged expansion of Vγ2Vδ2 T cells without significant side effects or anergy induction. These studies provide proof-of-principle that metabolic engineering can be used to derive live bacterial vaccines that boost Vγ2Vδ2 T cell immunity. Similar engineering of metabolic pathways to produce lipid Ags or B vitamin metabolite Ags could be used to derive live bacterial vaccine for other unconventional T cells that recognize nonpeptide Ags.
    Journal of immunology (Baltimore, Md. : 1950). 06/2014;
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    ABSTRACT: A cyclolavandulyl group is a C10 monoterpene with a branched and cyclized carbon skeleton. This monoterpene is rarely found in nature, and its biosynthesis is poorly understood. To determine the biosynthesis mechanism of this monoterpene, we sequenced the genome of Streptomyces sp. CL190, which produces lavanducyanin, a phenazine with an N-linked cyclolavandulyl structure. Sequencing and homology searches identified one candidate gene product that consists of only a cis-isoprenyl diphosphate synthase domain. Disruption of the gene and biochemical analysis of the recombinant enzyme demonstrated that the enzyme synthesized a cyclolavandulyl diphosphate essential for the biosynthesis of lavanducyanin. This enzyme is an unprecedented terpene synthase that catalyzes both the condensation of the C5 isoprene units and subsequent cyclization to form the cyclolavandulyl monoterpene structure.
    Journal of the American Chemical Society 03/2014; · 10.68 Impact Factor
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    ABSTRACT: Two new acyloin compounds were isolated from the thermophilic bacterium Thermosporothrix hazakensis SK20-1(T) . Genome sequencing of the bacterium and biochemical studies identified the thiamine diphosphate (TPP)-dependent enzyme Thzk0150, which is involved in the formation of acyloin. Through extensive analysis of the Thzk0150-catalyzed reaction products, we propose a putative reaction mechanism involving two substrates: 4-methyl-2-oxovalerate as an acyl donor and phenyl pyruvate as an acyl acceptor.
    ChemBioChem 01/2014; · 3.74 Impact Factor
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    Dataset: b817312e
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    ABSTRACT: The cytochrome P450 RauA from Rhodococcus erythropolis JCM 6824 catalyzes the hydroxylation of a nitrogen atom in the quinolone ring of aurachin, thereby conferring strong antibiotic activity on the aurachin alkaloid. Here, we report the crystal structure of RauA in complex with its substrate, a biosynthetic intermediate of aurachin RE. Clear electron density showed that the quinolone ring is oriented parallel to the porphyrin plane of the heme cofactor, while the farnesyl chain curls into a U-shape topology and is buried inside the solvent-inaccessible hydrophobic interior of RauA. The nearest atom from the heme iron is the quinolone nitrogen (4.3 Å), which is consistent with RauA catalyzing the N-hydroxylation of the quinolone ring to produce mature aurachin RE.
    FEBS letters 11/2013; · 3.54 Impact Factor
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    ABSTRACT: Thermus thermophilus exhibits hypersensitivity to a lysine analog, (S)-2-aminoethyl-cysteine (AEC). Cosmid libraries were constructed using genomes from two AEC-resistant mutants, AT10 and AT14, and the cosmids that conferred AEC resistance on the wild-type strain were isolated. When the cosmid library for mutant AT14 was screened, two independent cosmids conferring AEC resistance to the wild type were obtained. Two cosmids carried a common genomic region from TTC0795 to TTC0810. This region contains genes encoding two different ATP-binding cassette (ABC) transporters: one consisting of TTC0806/TTC0795 and the other consisting of TTC0967/TTC0968/TTC0969/TTC0970 using TTC0807 and TTC0966 as the periplasmic substrate-binding protein, respectively. Sequencing revealed that AT14 carries mutations in TTC0795 and TTC0969, causing decreases in the thermostability of the products. By similar screening for cosmids constructed for the mutant AT10, mutations were found at TTC0807 and TTC0969. Mutation in either of the transporter components gave partial resistance to AEC in the wild-type strain, while mutations of both transporters conferred complete AEC resistance. This result indicates that both transporters are involved in AEC uptake in T. thermophilus. To elucidate the mechanism of AEC uptake, crystal structures of TTC0807 were determined in several substrate-binding forms. The structures revealed that TTC0807 recognizes various basic amino acids by changing the side-chain conformation of Glu19, which interacts with the side-chain amino groups of the substrates.
    Journal of bacteriology 06/2013; · 3.94 Impact Factor
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    ABSTRACT: Aurachin RE is a prenylated quinoline antibiotic that was first isolated from the genus Rhodococcus. It shows potent antibacterial activity against a variety of Gram-positive bacteria. Here we have identified a minimal biosynthesis gene cluster for aurachin RE in Rhodococcus erythropolis JCM 6824 by using random transposon mutagenesis and heterologous production. The Rhodococcus aurachin (rau) gene cluster consists of genes encoding cytochrome P450 (rauA), prenyltransferase, polyketide synthase, and farnesyl pyrophosphate synthase, as well as others including genes involved in regulation and transport. Markerless gene disruption of rauA resulted in the complete loss of aurachin RE production and in the accumulation of a new aurachin derivative lacking the N-hydroxy group. When the recombinant RauA was expressed in Escherichia coli, it catalyzed N-hydroxylation of the derivative to form aurachin RE. This study establishes the biosynthetic pathway of aurachin RE and provides experimental evidence for the role of P450 RauA in catalyzing N-hydroxylation of the quinoline ring, which is indispensable for the antibacterial activity of aurachin RE.
    ChemBioChem 05/2013; · 3.74 Impact Factor
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    ABSTRACT: N-(2-Chlorobenzyl)-substituted hydroxamate, readily produced by hydrolysis of ketoclomazone, was identified as an inhibitor of 1-deoxy-d-xylulose 5-phosphate synthase (DXS), with an IC50 value of 1.0 μM. The compound inhibited the growth of Haemophilus influenzae. A convenient spectroscopic method for assaying DXS using NADPH-lactate dehydrogenase (LDH) is also reported.
    Chemical Communications 03/2013; · 6.38 Impact Factor
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    ABSTRACT: LysW has been identified as a carrier protein in the lysine biosynthetic pathway that is active through the conversion of α-aminoadipate (AAA) to lysine. In this study, we found that the hyperthermophilic archaeon, Sulfolobus acidocaldarius, not only biosynthesizes lysine through LysW-mediated protection of AAA but also uses LysW to protect the amino group of glutamate in arginine biosynthesis. In this archaeon, after LysW modification, AAA and glutamate are converted to lysine and ornithine, respectively, by a single set of enzymes with dual functions. The crystal structure of ArgX, the enzyme responsible for modification and protection of the amino moiety of glutamate with LysW, was determined in complex with LysW. Structural comparison and enzymatic characterization using Sulfolobus LysX, Sulfolobus ArgX and Thermus LysX identify the amino acid motif responsible for substrate discrimination between AAA and glutamate. Phylogenetic analysis reveals that gene duplication events at different stages of evolution led to ArgX and LysX.
    Nature Chemical Biology 02/2013; · 12.95 Impact Factor
  • Taro Ozaki, Makoto Nishiyama, Tomohisa Kuzuyama
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    ABSTRACT: The characterization of potential gene clusters is a promising strategy for the identification of novel natural products and the expansion of structural diversity. However, there are often difficulties in identifying potential metabolites because their biosynthetic genes are either silenced or expressed only at a low level. Here, we report the identification of a novel metabolite that is synthesized by a potential gene cluster containing an indole prenyltransferase gene (SCO7467) and a flavin-dependent monooxygenase (FMO) gene (SCO7468), which were mined from the genome of Streptomyces coelicolor A3(2). We introduced these two genes into the closely related Streptomyces lividans TK23 and analyzed the culture broths of the transformants. This process allowed us to identify a novel metabolite, 5-dimethylallylindole-3-acetonitrile (5-DMAIAN) that was overproduced in the transformant. Biochemical characterization of the recombinant SCO7467 and SCO7468 demonstrated the novel L-tryptophan metabolism leading to 5-DMAIAN. SCO7467 catalyzes the prenylation of L-tryptophan to form 5-dimethylallyl-L-tryptophan (5-DMAT). This enzyme is the first actinomycetes prenyltransferase known to catalyze the addition of a dimethylallyl group to the C-5 of tryptophan. SCO7468 then catalyzes the conversion of 5-DMAT into 5-dimethylallylindole-3-acetaldoxime (5-DMAIAOx). An aldoxime forming reaction catalyzed by the FMO enzyme was also identified for the first time in this study. Finally, dehydration of 5-DMAIAOx presumably occurs to yield 5-DMAIAN. This study provides insight into the biosynthesis of prenylated indoles that have been purified from actinomycetes.
    Journal of Biological Chemistry 02/2013; · 4.65 Impact Factor
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    ABSTRACT: Digging up skeletons: We report the identification and the functional characterization of two terpene cyclases (DtcycA and DtcycB) that were mined from the genome of Streptomyces sp. SANK 60404. DtcycA and DtcycB are novel bacterial diterpene cyclases for the synthesis of the cembrane skeleton.
    ChemBioChem 02/2013; 14(3):316-21. · 3.74 Impact Factor
  • Shota Isogai, Makoto Nishiyama, Tomohisa Kuzuyama
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    ABSTRACT: Furaquinocin is a natural polyketide-isoprenoid hybrid (meroterpenoid) produced by Streptomyces sp. strain KO-3988. All of the fur genes required for furaquinocin biosynthesis have been cloned, and the heterologous production of furaquinocin has been demonstrated in Streptomyces albus. Here, we report the identification of 8-amino-2,5,7-trihydroxynaphthalene-1,4-dione (8-amino-flaviolin) produced by the S. albus heterologous expression of the three contiguous genes encoding type III polyketide synthase (Fur1), monooxygenase (Fur2), and aminotransferase (Fur3) in the furaquinocin biosynthetic gene cluster. An S. albus transformant (S. albus/pWHM-Fur2_del3) harboring the fur gene cluster and lacking the fur3 gene did not produce furaquinocin, whereas furaquinocin production was restored when 8-amino-flaviolin was added to the culture medium of S. albus/pWHM-Fur2_del3. These results demonstrate that Fur3 aminotransferase is essential for furaquinocin biosynthesis and that 8-amino-flaviolin is an early-stage intermediate in furaquinocin biosynthesis. We propose that the biosynthetic pathway generating 8-amino-flaviolin is the common route for the biosynthesis of Streptomyces meroterpenoids.
    Bioorganic & medicinal chemistry letters 07/2012; 22(18):5823-6. · 2.65 Impact Factor
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    ABSTRACT: Fosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production in Pseudomonas syringae PB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that in Streptomyces fradiae and Streptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome of P. syringae PB-5123 lacks a gene encoding the PnPy decarboxylase found in the Streptomyces strains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin in Pseudomonas aeruginosa from a fosmid encoding the fosfomycin biosynthetic cluster from P. syringae PB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.
    Antimicrobial Agents and Chemotherapy 05/2012; 56(8):4175-83. · 4.57 Impact Factor
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    ABSTRACT: Green algae exclusively use the methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isoprenoids. The first enzyme of this pathway is 1-deoxy-D-xylulose 5-phosphate synthase (DXS, EC 2.2.1.7). Green algae have been thought to possess only a single DXS, in contrast to land plants, which have at least two isoforms that serve different roles in metabolism. The green microalga Botryococcus braunii has an extraordinary isoprenoid metabolism, as it produces large amounts of triterpene hydrocarbons. Here, we did cDNA cloning of DXSs from B. braunii and examined enzyme activities of the heterologously expressed proteins. Three distinct DXS isoforms were identified, all of which were functional and had similar kinetic properties, whereas the temperature dependence of enzyme activity showed considerable differences. Transcription of the genes was examined by real time quantitative RT-PCR. The three DXS genes were simultaneously expressed, and the expression levels were highest on day six after subculturing. B. braunii is the first green microalga demonstrated to have multiple DXS isoforms like land plants. This difference to other microalgae seems to mirror its special needs for extensive triterpene production by increasing the metabolic flow through the MEP pathway.
    Plant Science 04/2012; 185-186:309-20. · 2.92 Impact Factor
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    Tomohisa Kuzuyama, Haruo Seto
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    ABSTRACT: Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP.
    Proceedings of the Japan Academy Ser B Physical and Biological Sciences 01/2012; 88(3):41-52. · 2.77 Impact Factor
  • Tohru Dairi, Tomohisa Kuzuyama, Makoto Nishiyama, Isao Fujii
    ChemInform 11/2011; 42(45).
  • Takeo Tomita, Tomohisa Kuzuyama, Makoto Nishiyama
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    ABSTRACT: Glutamate dehydrogenase (GDH) catalyzes reversible conversion between glutamate and 2-oxoglutarate using NAD(P)(H) as a coenzyme. Although mammalian GDH is regulated by GTP through the antenna domain, little is known about the mechanism of allosteric activation by leucine. An extremely thermophilic bacterium, Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA (regulatory subunit) and GdhB (catalytic subunit). T. thermophilus GDH is unique in that the enzyme is subject to allosteric activation by leucine. To elucidate the structural basis for leucine-induced allosteric activation of GDH, we determined the crystal structures of the GdhB-Glu and GdhA-GdhB-Leu complexes at 2.1 and 2.6 Å resolution, respectively. The GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits. The GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex. Consistent with the structure, replacement of the amino acid residues of T. thermophilus GDH responsible for leucine binding made T. thermophilus GDH insensitive to leucine. Equivalent amino acid replacement caused a similar loss of sensitivity to leucine in human GDH2, suggesting that human GDH2 also uses the same allosteric site for regulation by leucine.
    Journal of Biological Chemistry 09/2011; 286(43):37406-13. · 4.65 Impact Factor
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    ABSTRACT: Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.
    Metabolic Engineering 08/2011; 13(6):629-37. · 6.86 Impact Factor

Publication Stats

2k Citations
459.51 Total Impact Points

Institutions

  • 1992–2014
    • The University of Tokyo
      • • Center for Biotechnology Research
      • • Institute of Molecular and Cellular Biosciences
      Edo, Tōkyō, Japan
  • 2011
    • Japan Women's University
      Edo, Tōkyō, Japan
  • 2010
    • Gunma National College of Technology
      Maebashi, Gunma Prefecture, Japan
  • 2006–2009
    • Seoul National University
      • • Research Institute for Agriculture and Life Science
      • • Department of Agricultural Biotechnology
      Seoul, Seoul, South Korea
    • Kyung Hee University
      Sŏul, Seoul, South Korea
  • 2007
    • Tokyo University of Agriculture
      • Department of Bioscience
      Tokyo, Tokyo-to, Japan
  • 2000–2006
    • Toyama Prefectural University
      • Biotechnology Research Center
      Toyama-shi, Toyama-ken, Japan
  • 2005
    • Salk Institute
      La Jolla, California, United States
    • University of Iowa
      Iowa City, Iowa, United States
  • 2001
    • Tokyo Institute of Technology
      • Chemistry Department
      Tokyo, Tokyo-to, Japan