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Engineering the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans for the kinetic resolution of Vince lactam.

Engineering the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans for the kinetic resolution of Vince lactam.
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Gao S, Zhu S, Huang R, Li H, Wang H, Zheng G,


Gao S, Zhu S, Huang R, Li H, Wang H, Zheng G, (click to view)

Gao S, Zhu S, Huang R, Li H, Wang H, Zheng G,

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Applied and environmental microbiology 2017 10 20() pii 10.1128/AEM.01780-17

Abstract

To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam were improved. Enantiomerically pure (-)-Vince lactam is the key synthon in the synthesis of antiviral drugs such as carbovir and abacavir, which are used to fight against human HIV and hepatitis B viruses. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give the (-)-Vince lactam with 99.2% (E >200) ee and 99.5% ee (E >200), respectively. To improve the thermostability of the enzyme, eleven residues with high B-factors calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value >200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam.Importance Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds of prominent medicinal relevance. Many studies about using (+)-γ-lactamase as biocatalyst for the desired resolution of the lactam have been reported. The efficiency of the native enzymes could be improved by using protein engineering methods such as directed evolution and rational design. Directed evolution makes it possible to engineer enzymes’ catalytic profiles including activity, enantioselectivity, substrate scope, and thermostability, whereas, the issue of optimizing multiproperties remains a central challenge. Compared to directed evolution, rational design or semi-rational designs are more efficient. The changes in amino acids are preconceived based on a detailed knowledge of protein structure, function and mechanism. This technology displays strong promise for optimizing the target properties for applications.In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase identified from Microbacterium hydrocarbonoxydans were tackled by semi-rational design. The protein engineering was initialized by using the strategy called combinatorial active-site saturation test to improve the enantioselectivity. At the same time two strategies were applied to identify mutation candidates to enhance the thermostability based on our calculations from both a static (B-FITTER based on the crystal structure) and a dynamic (RMSF values were calculated based on MD simulations) way. By conducting the screening in parallel we could achieve mutants with single enhanced property efficiently. In addition, it is also a fast way to validate if the design for improving each property works well. After combing the mutants with enhanced enantioselectivity and the mutants with improved thermostability, we successfully obtained the final mutants showing better properties in both enantioselectivity and thermostability. The engineered (+)-lactamase could be a good candidate as enantioselective catalyst for the preparation of enantiomerically pure Vince lactam.

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