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链霉菌590来源的L-甲硫氨酸脱羧酶的基因克隆、重组表达、纯化及特性分析

Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

作者信息

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

机构信息

Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan.

Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan.

出版信息

J Biochem. 2017 Apr 1;161(4):389-398. doi: 10.1093/jb/mvw083.

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.

摘要

来自链霉菌属590的L-蛋氨酸脱羧酶(MetDC)依赖于磷酸吡哆醛,催化L-蛋氨酸的非氧化脱羧反应,生成3-甲基硫丙胺和二氧化碳。经测定,MetDC基因(mdc)由1674个碱基对组成,编码557个氨基酸,其氨基酸序列与链霉菌属菌株的L-组氨酸脱羧酶和L-缬氨酸脱羧酶的氨基酸序列相似。克隆了mdc基因,并通过大肠杆菌异源表达重组MetDC。通过DEAE- Toyopearl和Ni-NTA琼脂糖柱层析对重组MetDC进行纯化。重组酶为同型二聚体,分子量为61000 Da,在45至55℃和pH 6.6时表现出最佳活性,在30℃以下和pH 4.6至7.0之间具有稳定性。L-蛋氨酸和L-正亮氨酸是MetDC的良好底物。L-蛋氨酸和L-正亮氨酸的米氏常数分别为30 mM和73 mM。重组MetDC(0.50 U/ml)能严重抑制人肿瘤细胞A431(表皮样卵巢癌细胞系)和MDA-MB-231(乳腺癌细胞系)的生长,但对人正常细胞NHDF-Neo(新生儿包皮真皮成纤维细胞系)的细胞毒性相对较低。本研究首次揭示了MetDC的基因特性和蛋白质序列。

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