Gene cloning, recombinant expression, purification and characterization of L-methionine decarboxylase from Streptomyces sp. 590.
Gene cloning, recombinant expression, purification and characterization of L-methionine decarboxylase from Streptomyces sp. 590.
复制标题
链霉菌 L-蛋氨酸脱羧酶基因克隆、重组表达、纯化和表征。
DOI：
10.1093/jb/mvw083
复制
发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Inagaki K.
中科院分区：
文献类型：
--
作者：
Hayashi M;Okada A;Yamamoto K;Okugochi T;Kusaka C;Kudou D;Nemoto M;Inagaki J;Hirose Yuu;Okajima T;Tamura T;Soda K;Inagaki K.
链霉菌L-甲硫氨酸脱羧酶（MetDC）590依赖于5′-磷酸吡哆醛，催化甲硫氨酸的非氧化脱羧反应，生成3-甲硫基丙胺和二氧化碳。MetDC基因（mdc）全长1,674 bp，编码557个氨基酸，其氨基酸序列与链霉菌的组氨酸脱羧酶和缬氨酸脱羧酶相似。菌株克隆并在大肠杆菌中异源表达了MetDC基因。经DEAE-Toyoptyline和Ni-NTA琼脂糖柱层析纯化重组MetDC。该重组酶为同源二聚体，分子量为61，000 Da，在45 ~ 55 °C和pH6.6范围内具有最佳活性，在30 °C以下和pH4.6 ~ 7.0范围内具有较好的稳定性。L-蛋氨酸和L-正亮氨酸的米氏常数分别为30和73 mM。重组MetDC（0.50 U/ml）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，然而对人正常细胞NHDF-Neo（来自新生儿包皮的真皮成纤维细胞系）显示相对低的细胞毒性。本研究首次揭示了MetDC的基因和蛋白质序列的性质。
l-Methionine decarboxylase (MetDC) fromStreptomycessp. 590 depends on pyridoxal 5′-phosphate and catalyzes the non-oxidative decarboxylation ofl-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 ofl-histidine decarboxylases andl-valine decarboxylases fromStreptomycessp. strains. Themdcgene was cloned and recombinant MetDC was heterologously expressed byEscherichia 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 andl-norleucine were good substrates for MetDC. The Michaelis constants forl-methionine andl-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.