Enhancing the Activity of LkTADH by Site-Directed Mutagenesis to Prepare Key Chiral Block of Statins

doi:10.13523/j.cb.20180909

China Biotechnology

2018, Vol. 38

Issue (9): 59-64 DOI: 10.13523/j.cb.20180909

Orginal Article

Enhancing the Activity of LkTADH by Site-Directed Mutagenesis to Prepare Key Chiral Block of Statins

Fang CHEN,Gang XU,Li-rong YANG,Jian-ping WU(

)

College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China

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Abstract

(S)-tert-butyl-6-chloro-5-hydroxyl-3-oxohexanoate [(S)-CHOH] is the key chiral intermediate of statins. Asymmetric reduction of tert-butyl-6-chloro-3,5- dioxohexanoate (CDOH) to (S)-CHOH catalyzed by alcohol dehydrogenases is a promising method. Nevertheless, the main problems is the low catalytic activity towards CDOH. First an alcohol dehydrogenase LkTADH (A94T/F147L/L199H/A202L) was further studied by reverse mutation and key sites (147,202) had been identified. M_F147L-A202L was obtained, which demonstrated 1-fold improvement in specific activity over LkTADH. After applying saturation mutagenesis at these two sites, M_F147I-A202L was obtained with 1.47-fold improvement in specific activity over LkTADH. The specific activity reached 10.17U/mg, which is the highest level as reported. Through dynamic analysis and molecular docking, the effect of mutation sites on enzyme activity was further analyzed.

Key words： Alcohol dehydrogenase (S)-tert-butyl-6-chloro-5-hydroxyl-3-oxohexanoate Asymmetric synthesis Molecular modification Saturated mutation

Received: 09 March 2018 Published: 12 October 2018

Corresponding Authors: Jian-ping WU E-mail: wjp@zju.edu.cn

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Cite this article:

Fang CHEN,Gang XU,Li-rong YANG,Jian-ping WU. Enhancing the Activity of LkTADH by Site-Directed Mutagenesis to Prepare Key Chiral Block of Statins. China Biotechnology, 2018, 38(9): 59-64.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180909 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I9/59

Fig.1 The biosynthesis route of (S)-CHOH

Table1 Enzyme activities of back mutants of LkTADH towards CDOH

Fig.2 Gene electrophoretogram of genomic DNA from mutantsM: DNA marker; 1-10: Mutants genome from Table 1

Fig.3 SDS-PAGE analysis of mutants overpressed in the recombinant E.coli (a) and purified forms(b)(a) M: Protein marker; 1,3,5,7:Supernatant extract of LkTADH,M_F147L,M_A202L and M_F147L-A202L; 2,4,6,8:Sediment extract of LkTADH,M_F147L,M_A202L and M_F147L-A202L (b) M: Protein marker; 1-5:Purified LkTADH,M_F147L,M_A202L,M_F147L-A202L and M_F147I-A202L

Table2 The kinetics paramaters of mutants

Fig.4 Enzyme activities of saturation mutants of LkTADH(L147 and L202) towards CDOH

Fig.5 Representation of the interactions between CDOH and LkTADH by molecular docking.

Fig. 6 Locations of mutation sites of LkTADH (a) and M_F147I-A202L (b)

Fig.7 Locations of mutation sites 94 of LkTADH (a) and M_F147I-A202L (b) and interactions between enzymes and substrate CDOH


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