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Enhancement in the catalytic activity of Sulfolobus solfataricus P2 (+)-γ-lactamase by semi-rational design with the aid of a newly established high-throughput screening method

  • Shuaihua Gao
  • Yingxiu Lu
  • Yuanyuan Li
  • Rong Huang
  • Guojun Zheng
Biotechnologically relevant enzymes and proteins

Abstract

(−)-γ-Lactam ((−)-2-azabicyclo[2.2.1]hept-5-en-3-one) has attracted increasing attention as the chiral intermediate of carbocyclic nucleosides most of which serve as pharmaceutical agents such as anti-HIV/HBV drugs abacavir and carbovir. So far, developing in vitro (+)-γ-lactamase-mediated biotransformation has been one of the most efficient approaches for the production of (−)-γ-lactam. In this study, the catalytic activity of the (+)-γ-lactamase from Sulfolobus solfataricus P2 was engineered by semi-rational design. Molecular docking and molecular dynamics simulation were carried out to target the key positions relevant to catalytic activity. Nine amino acid residues were selected for site saturation mutagenesis. To expedite the screening process, a sensitive colorimetric high-throughput screening method was established based on the Rimini test which was originally applied to distinguish primary amines from secondary amines. The screening process resulted in the achievement of several efficient mutants: V203N, V203Q, I336H, I336R, and Y388H. Synergy effects led to four final mutants (V203N/I336R, V203N/Y388H, I336R/Y388H, and V203N/I336R/Y388H) with enhanced enzyme activity after the combination of positive single mutants. The best mutant V203N/Y388H/I336R displayed a 21-fold higher enzyme efficiency (kcat/KM) compared to the wild-type enzyme. The result demonstrated that the biotransformation using the triple mutant as the catalyst reached > 49% conversion and > 99% enantiomeric excess at 80 °C after 2 h, which made it a good catalyst candidate to produce (−)-γ-lactam. The possible mechanism responsible for the improvement in the catalytic activity was explicated by analyzing the protein-ligand binding modes and interaction between the protein and the ligand.

Keywords

High-throughput screening γ-Lactam (+)-γ-lactamase Molecular docking Site-saturation mutagenesis Semi-rational design 

Notes

Funding information

This work was supported by the National Natural Science Foundation of China (NSFC, Grant No. 21706005), National Great Science and Technology Projects (2018ZX09721001), and the Fundamental Research Funds for the Central Universities (No. ZY1713).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9428_MOESM1_ESM.pdf (497 kb)
ESM 1 (PDF 497 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Chemical Resources EngineeringBeijing University of Chemical TechnologyBeijingPeople’s Republic of China
  2. 2.Department of ChemistryUniversity of CaliforniaBerkeleyUSA
  3. 3.California Institute for Quantitative BiosciencesUniversity of CaliforniaBerkeleyUSA

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