远藤黄诺卡氏菌 IFM10152 细胞色素 P450 单加氧酶 A 环对位羟化大豆苷元。

A-ring ortho-specific monohydroxylation of daidzein by cytochrome P450s of Nocardia farcinica IFM10152.

机构信息

School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics and Institute of Bioengineering, Seoul National University, Seoul, South Korea.

出版信息

Biotechnol J. 2009 Nov;4(11):1586-95. doi: 10.1002/biot.200900157.

DOI:10.1002/biot.200900157

PMID:19918785

Abstract

The bioconversion of the isoflavonoid daidzein using whole cell Nocardia farcinica IFM10152 showed two kinds of major metabolic modifications, i.e. mono-hydroxylation and subsequent O-methylation. The major hydroxylated products of daidzein prior to the O-methylation reaction were 3',4',7-trihydroxyisoflavone (3'-ODI), 4',6,7-trihydroxyisoflavone (6-ODI) and 4',7,8-trihydroxyisoflavone (8-ODI), which are mono-hydroxylated at the ortho position of each hydroxyl group of daidzein. To identify monooxygenases playing a key role in the monohydroxylation of the A-ring of daidzein, all genes of 27 cytochrome P450s from N. farcinica IFM10152 were cloned and transformed into a E. coli BL21 (DE3) host system. By this enzymatic reaction using the mutants and the genome sequence analysis of N. farcinica IFM10152, it was revealed that nfa12130 and nfa33880 P450 genes clustered with their own ferredoxins and ferredoxin reductases (nfa12140+nfa12150 and nfa338870+nfa33860, respectively) are responsible for the hydroxylation of the A-ring of daidzein, and their major reaction products were 6-ODI and 8-ODI, respectively.

摘要

利用全细胞诺卡氏菌 IFM10152 将异黄酮染料木素进行生物转化，显示出两种主要的代谢修饰，即单羟基化和随后的 O-甲基化。在 O-甲基化反应之前，染料木素的主要羟基化产物是 3'，4'，7-三羟基异黄酮（3'-ODI），4'，6，7-三羟基异黄酮（6-ODI）和 4'，7，8-三羟基异黄酮（8-ODI），它们在染料木素每个羟基的邻位发生单羟基化。为了鉴定在染料木素 A 环单羟基化中起关键作用的单加氧酶，从诺卡氏菌 IFM10152 中克隆了所有 27 种细胞色素 P450 的基因，并转化到大肠杆菌 BL21（DE3）宿主系统中。通过使用突变体的这种酶促反应和对 N. farcinica IFM10152 的基因组序列分析，揭示了 nfa12130 和 nfa33880 P450 基因与它们各自的铁氧还蛋白和铁氧还蛋白还原酶（nfa12140+nfa12150 和 nfa338870+nfa33860）聚类，负责染料木素 A 环的羟基化，其主要反应产物分别为 6-ODI 和 8-ODI。