A triple mutant improving activity of cytochrome P450 BM-3 to catalyze indole hydroxylation：D168L/E435T/V445A

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

doi:10.3969/j.issn.0438-1157.2014.04.030

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A triple mutant improving activity of cytochrome P450 BM-3 to catalyze indole hydroxylation：D168L/E435T/V445A

更新时间：2025-12-10

- A triple mutant improving activity of cytochrome P450 BM-3 to catalyze indole hydroxylation：D168L/E435T/V445A
- CIESC Journal Issue 4, Pages: 1374-1380(2014)
- 作者机构：
  
  1. 浙江大学生物工程研究所,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
- 作者简介：
- 基金信息：
- DOI：10.3969/j.issn.0438-1157.2014.04.030
  CLC： Q962.2
- Online First：30 October 2013，
  
  Published：30 October 2013
- 稿件说明：
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ZHANG Pengpai, HU Sheng, MEI Lehe, et al. A triple mutant improving activity of cytochrome P450 BM-3 to catalyze indole hydroxylation：D168L/E435T/V445A[J]. CIESC Journal, 2014, (4): 1374-1380.
DOI：

ZHANG Pengpai, HU Sheng, MEI Lehe, et al. A triple mutant improving activity of cytochrome P450 BM-3 to catalyze indole hydroxylation：D168L/E435T/V445A[J]. CIESC Journal, 2014, (4): 1374-1380. DOI： 10.3969/j.issn.0438-1157.2014.04.030.

摘要

为进一步提高P450 BM-3催化吲哚合成靛蓝的能力，在前期获得的突变酶D168L/E435T基础上引入V445A突变，获得突变酶D168L/E435T/V445A，该突变酶与D168L/E435T及V445A相比，对吲哚的亲和力分别降低41.5%及35.8%，转化数（kcat）分别提高1.61倍及1.31倍，催化效率（kcatKm-1）分别提高1.53倍及1.47倍；该突变酶对电子的耦合率也提高至24.7%，较D168L/E435T提高39.5%，较V445A提高64.7%，说明该突变酶在电子的利用率上有明显改善；此外，该突变酶催化副产物靛玉红的比例大幅降低，降低为D168L/E435T的41.7%及V445A的30.6%，说明该突变酶催化吲哚的区域选择性上也更加有利于靛蓝形成。

Abstract

To further enhance hydroxylation ability with indole of P450 BM-3

site-directed mutagenesis with V445A was introduced to the mutant D168L/E435T obtained in earlier research

and mutant D168L/E435T/V445A was obtained. Compared with D168L/E435T and V445A

mutant D168L/E435T/V445A exhibited lower affinity with indole

but higher improved turnover rate (kcat). Correspondingly

catalytic efficiency (kcatKm-1) was increased to 1.53 times and 1.47 times respectively. The electronic coupling rate of the mutant was also improved by 24.7%

39.5% higher than that of D168L/E435T and 64.7% higher than that of V445A

showing better efficiency of electronic utilization of this new mutant enzyme. Mutant D168L/E435T/V445A also displayed improved regioselectivity with indole in indigo synthesis

with the proportion of by-product indirubin 41.7% of that of D168L/E435T and 30.6% of that of V445A.

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