Improving indole-hydroxylation ability of cytochrome P450 BM-3 by site-directed mutagenesis

ZHANG Pengpai; HU Sheng; HUANG Jun; MEI Lehe; LEI Yinlin; JIN Zhihua; YAO Shanjing

doi:10.3969/j.issn.0438-1157.2013.09.035

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Improving indole-hydroxylation ability of cytochrome P450 BM-3 by site-directed mutagenesis

更新时间：2025-12-10

- Improving indole-hydroxylation ability of cytochrome P450 BM-3 by site-directed mutagenesis
- CIESC Journal Issue 9, Pages: 3331-3337(2013)
- 作者机构：
  
  1. 浙江大学化学工程与生物工程学系,浙江,杭州,310027
  2. 浙江大学宁波理工学院生物与化学工程学院,浙江,宁波,315100
  3. 浙江科技学院生物与化学工程学院,浙江,杭州,310023
- 作者简介：
- 基金信息：
- DOI：10.3969/j.issn.0438-1157.2013.09.035
  CLC： Q962.2
- Online First：05 September 2013，
  
  Published：05 September 2013
- 稿件说明：
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ZHANG Pengpai, HU Sheng, HUANG Jun, et al. Improving indole-hydroxylation ability of cytochrome P450 BM-3 by site-directed mutagenesis[J]. CIESC Journal, 2013, (9): 3331-3337.
DOI：

ZHANG Pengpai, HU Sheng, HUANG Jun, et al. Improving indole-hydroxylation ability of cytochrome P450 BM-3 by site-directed mutagenesis[J]. CIESC Journal, 2013, (9): 3331-3337. DOI： 10.3969/j.issn.0438-1157.2013.09.035.

摘要

为进一步提高细胞色素P450 BM-3(A74G/F87V/L188Q)对吲哚的羟基化能力

根据酶结构与功能的关系

以突变酶E435T为基础

在168位点引入D168L突变

获得了吲哚羟基化能力得到显著提高的突变酶D168L/E435T。突变酶对吲哚的Km为1.72 mmol·L-1(父本2.09 mmol·L-1)

转化数(kcat)为28.15 min-1(父本4.04 min-1)

表明D168L定点突变可以略微提高酶对底物的亲和力

但主要的效应是促进了底物的转化速率

这两个效应的综合表现是使酶的催化效率(kcat Km-1)比父本酶提高了8.48倍。此外

产物中副产物靛玉红的比例也降低为1.2%(父本7.3%)

这说明该突变酶催化吲哚的区域选择性上也更有利于靛蓝的生成。

Abstract

According to the relationship between the structure and function of enzyme

it was speculated that the combination of the mutant enzyme D168L and E435T might improve the catalytic ability of indole to generate indigo.The mutant enzyme D168L/E435T was produced by site-directed mutagenesis conducted on the 168th site of the P450 BM-3(A74G/F87V/L188Q/E435T)and it was identified with higher hydroxylation activity toward indole in comparison to its parent enzyme.Concomitantly

its Km for indole was 1.72 mmol·L-1 and this value was lower compared to its parent enzyme(2.09 mmol·L-1).Also with the kcat=28.15 min-1

the turnover rate of the mutant enzyme was increasing up to 6.97-fold that of its parent enzyme(4.04 min-1).The catalytic efficiency(kcatKm-1)was correspondingly raised to 8.48 times as much as that of its parent enzyme.Moreover

the proportion of the byproducts indirubin reduced to 1.2%

much lower than that of its parent mutant(7.3%)

which indicated that this regioselectivity of mutant enzyme to catalyze indole was more conducive to generating indigo.

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Related Institution

Institute of Bioengineering, Zhejiang University

School of Biological and Chemical Engineering, Ningbo Institute of Technology

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