Parry R J, Li W
Department of Chemistry, Rice University, Houston, Texas 77005-1892, USA.
J Biol Chem. 1997 Sep 12;272(37):23303-11. doi: 10.1074/jbc.272.37.23303.
The valanimycin producer Streptomyces viridifaciens 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.
缬氨霉素产生菌绿色产色链霉菌含有一个双组分酶系统，该系统催化异丁胺氧化为异丁羟胺。该酶系统的一个组分为异丁胺羟化酶，另一个组分为黄素还原酶。通过染色体步移法从绿色产色链霉菌中克隆到了异丁胺羟化酶所需的黄素还原酶编码基因（vlmR）。该基因编码一个由194个氨基酸组成的蛋白质，计算分子量为21265 Da，计算pI为10.2。将vlmR基因作为N端His标签衍生物在大肠杆菌中过表达，产生了一种可溶蛋白，并将其纯化至均一。通过凝血酶切割从过表达蛋白上去除N端His标签，也产生了一种可溶蛋白。这两种形式的蛋白均表现出高度的黄素还原酶活性，且凝血酶切割形式的蛋白与异丁胺羟化酶协同作用，催化异丁胺转化为异丁羟胺。动力学数据表明，过表达的蛋白优先利用FAD和NADPH，而不是FMN、核黄素和NADH。VlmR蛋白推导的氨基酸序列与其他几种黄素还原酶具有相似性，可能构成一个新的黄素还原酶家族。
