title: Recombinant expression, molecular characterization and crystal structure of antitumor enzyme, L-lysine α-oxidase from Trichoderma viride.
abstract: L-Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form α-keto-ε-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 Å 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.
authors: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan; Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; and Enzyme Sensor Co., Ltd., Tsukuba, Ibaraki 305-0047, Japan.
authors: Marie
authors: M
authors: Amano
authors: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan; Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; and Enzyme Sensor Co., Ltd., Tsukuba, Ibaraki 305-0047, Japan.
authors: Haruka
authors: H
authors: Mizuguchi
authors: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan; Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; and Enzyme Sensor Co., Ltd., Tsukuba, Ibaraki 305-0047, Japan.
authors: Tadahisa
authors: T
authors: Sano
authors: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan; Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; and Enzyme Sensor Co., Ltd., Tsukuba, Ibaraki 305-0047, Japan.
authors: Hiroki
authors: H
authors: Kondo
authors: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan; Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; and Enzyme Sensor Co., Ltd., Tsukuba, Ibaraki 305-0047, Japan.
authors: Kengo
authors: K
authors: Shinyashiki
authors: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan; Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; and Enzyme Sensor Co., Ltd., Tsukuba, Ibaraki 305-0047, Japan.
authors: Junko
authors: J
authors: Inagaki
authors: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan; Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; and Enzyme Sensor Co., Ltd., Tsukuba, Ibaraki 305-0047, Japan.
authors: Takashi
authors: T
authors: Tamura
authors: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan; Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; and Enzyme Sensor Co., Ltd., Tsukuba, Ibaraki 305-0047, Japan.
authors: Tatsuya
authors: T
authors: Kawaguchi
authors: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan; Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; and Enzyme Sensor Co., Ltd., Tsukuba, Ibaraki 305-0047, Japan.
authors: Hitoshi
authors: H
authors: Kusakabe
authors: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan; Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; and Enzyme Sensor Co., Ltd., Tsukuba, Ibaraki 305-0047, Japan.
authors: Katsumi
authors: K
authors: Imada
authors: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan; Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan; and Enzyme Sensor Co., Ltd., Tsukuba, Ibaraki 305-0047, Japan.
authors: Kenji
authors: K
authors: Inagaki
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D048170:Streptomyces lividans / Q000235:genetics; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D000970:Antineoplastic Agents; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C041123:L-lysine oxidase
keywords: L-lysine α-oxidase; antitumor enzyme; cDNA cloning; crystal structure; recombinant expression