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.
| Original language | English |
|---|---|
| Pages (from-to) | 389-398 |
| Number of pages | 10 |
| Journal | Journal of Biochemistry |
| Volume | 161 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - Apr 1 2017 |
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Keywords
- antitumour enzyme
- gene cloning
- l-methionine decarboxylase
- pyridoxal 5′-phosphate
- recombinant expression
ASJC Scopus subject areas
- Medicine(all)
- Biochemistry
- Molecular Biology
Cite this
Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590. / Hayashi, Masaya; Okada, Akane; Yamamoto, Kumiko; Okugochi, Tomomi; Kusaka, Chika; Kudou, Daizou; Nemoto, Michiko; Inagaki, Junko; Hirose, Yuu; Okajima, Toshihide; Tamura, Takashi; Soda, Kenji; Inagaki, Kenji.
In: Journal of Biochemistry, Vol. 161, No. 4, 01.04.2017, p. 389-398.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590
AU - Hayashi, Masaya
AU - Okada, Akane
AU - Yamamoto, Kumiko
AU - Okugochi, Tomomi
AU - Kusaka, Chika
AU - Kudou, Daizou
AU - Nemoto, Michiko
AU - Inagaki, Junko
AU - Hirose, Yuu
AU - Okajima, Toshihide
AU - Tamura, Takashi
AU - Soda, Kenji
AU - Inagaki, Kenji
PY - 2017/4/1
Y1 - 2017/4/1
N2 - 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.
AB - 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.
KW - antitumour enzyme
KW - gene cloning
KW - l-methionine decarboxylase
KW - pyridoxal 5′-phosphate
KW - recombinant expression
UR - http://www.scopus.com/inward/record.url?scp=85021849898&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85021849898&partnerID=8YFLogxK
U2 - 10.1093/jb/mvw083
DO - 10.1093/jb/mvw083
M3 - Article
C2 - 28003434
AN - SCOPUS:85021849898
VL - 161
SP - 389
EP - 398
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 4
ER -