An NADPH:FAD oxidoreductase from the valanimycin producer, Streptomyces viridifaciens - Cloning, analysis, and overexpression
An NADPH:FAD oxidoreductase from the valanimycin producer, Streptomyces viridifaciens - Cloning, analysis, and overexpression
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DOI：
10.1074/jbc.272.37.23303
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发表时间：
1997-09-12
影响因子：
4.8
通讯作者：
Li, WY
中科院分区：
文献类型：
--
作者：
Parry, RJ;Li, WY
缬氨霉素产生菌Streptomyces uilidifaciens含有催化异丁胺氧化为异丁基羟胺的双组分酶系统，其中一个组分是异丁胺羟化酶，另一个组分是黄素还原酶。该基因编码194个氨基酸的蛋白质，计算的质量为21，265 Da，计算的pI为10.2。在大肠杆菌中过表达vlmR基因作为N-末端His-标签衍生物，产生纯化至均一的可溶性蛋白质。通过凝血酶切割从过表达的蛋白质去除N-末端His-标签也产生可溶性蛋白质。两种形式的蛋白质都表现出高度的黄素还原酶活性，并且凝血酶切割的形式与异丁胺羟化酶组合起作用以催化异丁胺转化为异丁基羟胺。动力学数据表明过表达的蛋白质优先于FMN、核黄素、和NADH。推导的氨基酸序列的VlmR蛋白表现出相似的其他几个黄素还原酶，可能构成一个新的家庭黄素还原酶。
The valanimycin producer Streptomyces uilidifaciens contains a two-component enzyme system that catalyzes the oxidation of isobutylamine to isobutylhydroxylamine, One component of this enzyme system is isobutylamine hydroxylase, and the other component is a flavin reductase, The gene (vlmR) encoding the flavin reductase required by isobutylamine hydroxylase has been cloned from S. viridifaciens by chromosome walking, The gene codes for a protein of 194 amino acids with a calculated mass of 21,265 Da and a calculated pI of 10.2, Overexpression of the vlmR gene in Escherichia coli as an N-terminal His-tag derivative yielded a soluble protein that was purified to homogeneity. Removal of the N-terminal His-tag from the overexpressed protein by thrombin cleavage also produced a soluble protein, Both forms of the protein exhibited a high degree of flavin reductase activity, and the thrombin-cleaved form functioned in combination with isobutylamine hydroxylase to catalyze the conversion of isobutylamine to isobutylhydroxylamine, Kinetic data indicate that the overexpressed protein utilizes FAD and NADPH in preference to FMN, riboflavin, and NADH. The deduced amino acid sequence of the VlmR protein exhibited similarity to several other flavin reductases that may constitute a new family of flavin reductases.