title: Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.
abstract: l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.
authors.affiliation: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan.
authors.forename: Masaya
authors.initials: M
authors.lastname: Hayashi
authors.affiliation: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan.
authors.forename: Akane
authors.initials: A
authors.lastname: Okada
authors.affiliation: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Okugochi
authors.affiliation: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan.
authors.forename: Chika
authors.initials: C
authors.lastname: Kusaka
authors.affiliation: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan.
authors.forename: Daizou
authors.initials: D
authors.lastname: Kudou
authors.affiliation: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Nemoto
authors.affiliation: Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Inagaki
authors.affiliation: Department of Environmental and Life Sciences, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan.
authors.forename: Yuu
authors.initials: Y
authors.lastname: Hirose
authors.affiliation: Institute of Scientific and Industrial Research, Osaka University, 8-1 Miho-gaoka, Osaka 567-0047, Japan.
authors.forename: Toshihide
authors.initials: T
authors.lastname: Okajima
authors.affiliation: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tamura
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Soda
authors.affiliation: Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Inagaki
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002245:Carbon Dioxide / Q000378:metabolism; D002262:Carboxy-Lyases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010802:Phylogeny; D011437:Propylamines / Q000378:metabolism; D055503:Protein Multimerization; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011437:Propylamines; D011994:Recombinant Proteins; D002245:Carbon Dioxide; C055136:3-methylthiopropylamine; D011732:Pyridoxal Phosphate; D008715:Methionine; D002262:Carboxy-Lyases; C066836:methionine decarboxylase
keywords: antitumour enzyme; gene cloning; l-methionine decarboxylase; pyridoxal 5′-phosphate; recombinant expression