Faculty Profiles - TAMURA Takashi

Recombinant expression, molecular characterization and crystal structure of antitumor enzyme, l-lysine alpha-oxidase from Trichoderma viride* Reviewed

Reactivity of sorbose dehydrogenase from Sinorhizobium sp 97507 for 1,5-anhydro-D-glucitol Reviewed

Toshio Araki, Tomoko Nakatsuka, Fuminao Kobayashi, Emi Watanabe-Ishimaru, Hirokazu Sanada, Takashi Tamura, Kenji Inagaki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 79 ( 7 ) 1130 - 1132 2015.7

Marie Amano, Haruka Mizuguchi, Tadahisa Sano, Hiroki Kondo, Kengo Shinyashiki, Junko Inagaki, Takashi Tamura, Tatsuya Kawaguchi, Hitoshi Kusakabe, Katsumi Imada, Kenji Inagaki

JOURNAL OF BIOCHEMISTRY 157 ( 6 ) 549 - 559 2015.6

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

L-Lysine alpha-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form alpha-keto-epsilon-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 <remove> resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.

DOI： 10.1093/jb/mvv012

Scopus

Other Link： http://orcid.org/0000-0003-1342-8885

Draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin Reviewed

Kenshiro Oshima, Masahira Hattori, Hitomi Shimizu, Koji Fukuda, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

Genome Announcements 3 ( 4 ) e00715 2015

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD.

File： Microbiology Resource Announcements-2015-Oshima-e00715-15.full.pdf

DOI： 10.1128/genomeA.00715-15

Scopus

Sulfur Transport System for the formation of iron-sulfur cluster

Tamura Takashi, Asano Kaori

VITAMINS 89 ( 2 ) 80 - 82 2015

Mechanism of N_2 fixation and H_2 production by nitrogenase

Tamura Takashi

VITAMINS 89 ( 8 ) 409 - 412 2015

Glycerol kinase from Cellulomonas sp. NT3060 which has high resistance against preservatives Reviewed

Sogabe Atsushi, Kitabayashi Masao, Morishima Kenichi, Furukawa Miyoko, Hatta Takashi, Fukuda Yasuhisa, Nishise Hiroshi, Oka Masanori, Tamura Takashi, Inagaki Kenji

Seibutsu-kogaku Kaishi 92 ( 8 ) 402 - 409 2014.8

Language：Japanese Publisher：公益社団法人日本生物工学会

Glycerol kinase is suitable for clinical diagnostic use in levels of neutral fat. Glycerol kinase from Cellulomonas sp. NT3060 shows good stability against several preservatives such as N-methylisothiazolone and imidazolidinylurea. We have cloned glpK gene from Cellulomonas sp. NT3060. This glpK gene is composed of 1,518 bp nucleotides and encodes 505 amino acid residues. The glpK gene was expressed in Escherichia coli and Cellulomonas sp. glycerol kinase was purified to homogeneity by column chromatography. The glycerol kinase exhibited high tolerance to various preservatives compared to other glycerol kinases. This tolerance was found to be favorable feature as diagnostic reagent for quantity of triglyceride.

CiNii Article

CiNii Books

The Role of Amino Acid Residues in the Active Site of L-Methionine gamma-lyase from Pseudomonas putida Reviewed

Mitsuki Fukumoto, Daizou Kudou, Shouko Murano, Tomoo Shiba, Dan Sato, Takashi Tamura, Shigeharu Harada, Kenji Inagaki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76 ( 7 ) 1275 - 1284 2012.7

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine gamma-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel beta-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

DOI： 10.1271/bbb.110906

Arg305 of Streptomyces L-glutamate oxidase plays a crucial role for substrate recognition Reviewed

Tomohiro Utsumi, Jiro Arima, Chika Sakaguchi, Takashi Tamura, Chiduko Sasaki, Hitoshi Kusakabe, Shigetoshi Sugio, Kenji Inagaki

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 417 ( 3 ) 951 - 955 2012.1

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Recently, we have solved the crystal structure of L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 (PDB code: 2E1M), the substrate specificity of which is strict toward L-glutamate. By a docking simulation using.-glutamate and structure of LGOX, we selected three residues, Arg305, His312, and Trp564 as candidates of the residues associating with recognition of L-glutamate. The activity of LGOX toward L-glutamate was significantly reduced by substitution of selected residues with Ala. However, the enzyme, Arg305 of which was substituted with Ala, exhibited catalytic activity toward various L-amino acids. To investigate the role of Arg305 in substrate specificity, we constructed Arg305 variants of LGOX. In all mutants, the substrate specificity of LGOX was markedly changed by the mutation. The results of kinetics and pH dependence on activity indicate that Arg305 of LGOX is associated with the interaction of enzyme and side chain of substrate. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.12.033

Quantum Chemical Mechanism of Hydrogeanses

2PT109 Structure of L-lysine α-oxidase from Trichoderma viride(The 50th Annual Meeting of the Biophysical Society of Japan)

Sano Tadahisa, Kawaguchi Tatsuya, Amano Marie, Shinyashiki Kengo, Nakata Haruka, Tamura Takashi, Inagaki Kenji, Kusakabe Hitoshi, Imada Katsumi

Seibutsu Butsuri 52 S123 2012

Tamura Takashi

VITAMINS 86 ( 7 ) 412 - 414 2012

Selenite Reduction by the Thioredoxin System: Kinetics and Identification of Protein-Bound Selenide Reviewed

Takashi Tamura, Kumi Sato, Kentaro Komori, Takeshi Imai, Mitsuhiko Kuwahara, Takahiro Okugochi, Hisaaki Mihara, Nobuyoshi Esaxi, Kenji Inagaki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 ( 6 ) 1184 - 1187 2011.6

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

Selenite (SeO32-) assimilation into a bacterial selenoprotein depends on thioredoxin (trx) reductase in Esherichia coli, but the molecular mechanism has not been elucidated. The mineral-oil overlay method made it possible to carry out anaerobic enzyme assay, which demonstrated an initial lag-phase followed by time-dependent steady NADPH consumption with a positive cooperativity toward selenite and trx. SDS-PAGE/auto-radiography using Se-75-labeled selenite as substrate revealed the formation of trx-bound selenium in the reaction mixture. The protein-bound selenium has metabolic significance in being stabilized in the divalent state, and it also produced the selenopersulfide (-S-SeH) form by the catalysis of E. coli trx reductase (TrxB).

DOI： 10.1271/bbb.100847

CiNii Article

Identification and Conformer Analysis of a Novel Redox-Active Motif, Pro-Ala-Ser-Cys-Cys-Ser, in Drosophila Thioredoxin Reductase by Semiempirical Molecular Orbital Calculation Reviewed

Mitsuhiko Kuwahara, Takashi Tamura, Kentaro Kawamura, Kenji Inagaki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 ( 3 ) 516 - 521 2011.3

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

Mammalian thioredoxin reductases (TrxRs) contain selenium as selenocysteine (Sec) in the C-terminal redox center -Gly-Cys-Sec-Gly-OH to reduce Trx and other substrates; a Sec-to-Cys substitution in mammalian TrxR yields an almost inactive enzyme. The corresponding tetrapeptide sequence in Drosophila melanogaster TrxR (Dm-TrxR), -Ser-Cys-Cys-Ser-OH, endows the orthologous enzyme with a catalytic competence similar to mammalian selenoenzymes, but implementation of the Ser-containing tetrapeptide sequence SCCS into the mammalian enzyme does not restore the activity of the Sec-to-Cys mutant form (turnover number < 2/min). MOPAC calculation suggested that the C-terminal hexapeptide Pro-Ala-Ser-Cys-Cys-Ser-OH functions as a redox center that alleviates the necessity for selenium in Dm-TrxR, and a mutant form of human lung TrxR that mimics this hexapeptide sequence showed improved catalytic turnover (17.4/min for DTNB and 13.2/min for E. coli trx) compared to the Sec-to-Cys mutant. MOPAC calculation also suggested that the dominant form of the Pro-containing hexapeptide is a C+ conformation, which perhaps has a catalytic advantage in facile reduction of the intramolecular disulfide bond between Cys497 and Cys498 by the N-terminal redox center in the neighboring subunit.

DOI： 10.1271/bbb.100740