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Bocola, Marco

Chen, Haibin

Hayes, Marc

Huang, Shuai

Wang, Xiao-Lei

Xu, Junpeng

English

Engineering Conferences International

Monday, May 23 2022                                                          Session 1:  Enzyme Engineering in Synthetic Biology IN SILICO SCREENING OF TRANSAMINASE USING SEMI-EMPIRICAL QM/MM APPROACH  Haibin Chen, Enzymaster (Ningbo) Bio-Engineering Co., Ltd, USA haibin.chen@enzymaster.com  Shuai Huang, Shanghai Jiao Tong University, USA Junpeng Xu, Enzymaster (Ningbo) Bio-Engineering Co., Ltd, USA Marco Bocola, Enzymaster Deutschland GmbH, USA Xiao-Lei Wang, Shanghai Jiao Tong University USA  Key Words: Transaminase reaction mechanism, QM/MM calculation, in silico screening, computer-aided directed evolution of enzymes   Transaminases (TAs) are being widely used to produce chiral amines, and the demand of engineered TA variants to meet specific production processes is increasing. However, the engineering of TA by directed evolution methodology is still heavily relying on wet lab screening, which is often costly and time consuming. In this study we developed a reliable high throughput in silico (pre-)screening method for TA engineering.  We established an in silico model of the catalytic reactions of wild-type (WT) TA from Aspergillus fumigatus Af293 (AfTA), identified the rate limiting step of TA reaction, and performed in silico screening of a large number of AfTA variants using a new framework for computer-aided directed evolution of enzymes (CADEE) 1 with semi-empirical QM/MM approach in combination with DFT studies at B3LYP/6-31G-level for calibration.  Firstly, we used quantum mechanical/molecular mechanical (QM/MM) calculations to investigate the detailed catalytic reaction mechanism and the rate limiting step of PLP-dependent transaminase reaction for several chiral amines such as (R)-α-phenylethylamine (R-PEA) and (R)-1-naphthylethylamine (R-NEA). By calculating the proton at different positions on the amines (shown in figure 1 inset), we proposed a mechanism that differs from the one reported for a different TA in literature 2.   After validation of the energy profile of the reaction (shown in Figure 1) with WT AfTA, we performed in silico screening of AfTA variants with CADEE calculations and were able to identify activity-enhancing mutations in the active-site for R-PEA vs. R-NEA in good agreement with the experimental results of AfTA variants. In conclusion, our study demonstrated that CADEE with semi-empirical QM/MM approach can be used as a fast and accurate in silico (pre-)screening method for TA engineering.                       References:  (1)  Amrein, B. A.; Steffen-Munsberg, F.; Szeler, I.; Purg, M.; Kulkarni, Y.; Kamerlin, S. C. L. CADEE: Computer-Aided Directed Evolution of Enzymes. IUCrJ 2017, 4 (Pt 1), 50–64. https://doi.org/10.1107/S2052252516018017. (2)  Cassimjee, K. E.; Manta, B.; Himo, F. A Quantum Chemical Study of the ω-Transaminase Reaction Mechanism. Org. Biomol. Chem. 2015, 13 (31), 8453–8464. https://doi.org/10.1039/C5OB00690B Inset – The proton at different positions of the PLP-amine-IM Figure 1 – Rate limiting step of transamination reaction for R-PEA.  

In silico screening of transaminase using semi-empirical QM/MM approach

https://dc.engconfintl.org/cgi/viewcontent.cgi?filename=0&article=1004&context=enzyme_xxvi&type=additional

In silico screening of transaminase using semi-empirical QM/MM approach

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Engineering Conferences International