4.8 Article

Computationally Driven Rational Design of Substrate Promiscuity on Serine Ester Hydrolases

期刊

ACS CATALYSIS
卷 11, 期 6, 页码 3590-3601

出版社

AMER CHEMICAL SOC
DOI: 10.1021/acscatal.0c05015

关键词

enzymology; esterase; protein engineering; substrate promiscuity; computational chemistry

资金

  1. grant INMARE from the European Union's Horizon 2020 [634486]
  2. Marine Biotechnology ERANET [PCIN-2017-078]
  3. Spanish Ministry of Science and Innovation [BIO2017-85522-R, PID2019-106370RB-I00/AEI, FPU19/00608]
  4. Ministerio de Economia, Industria y Competitividad
  5. Ministerio de Ciencia, Innovacion y Universidades
  6. Agencia Estatal de Investigacion (AEI)
  7. Fondo Europeo de Desarrollo Regional (FEDER)
  8. European Union (EU)

向作者/读者索取更多资源

Through studying enzymes with narrow substrate spectra, it was demonstrated that active site parameters can be manipulated to alter substrate specificity and design new enzyme variants with broader substrate promiscuity.
Enzymes with a broad substrate specificity are of great interest both at the basic and applied level. Understanding the main parameters that make an enzyme substrate ambiguous could be thus important not only for their selection from the ever-increasing amount of sequencing data but also for engineering a more substrate promiscuous variant. This issue, which remains unresolved, was herein investigated by targeting a serine ester hydrolase (EH102), which exhibits a narrow substrate spectrum, being only capable of hydrolyzing 16 out of 96 esters tested. By using a modeling approach, we demonstrated that one can rationalize active site parameters defining substrate promiscuity, and that based on them the substrate specificity can be significantly altered. This was accomplished by designing two variants, EH102(DM2) and EH102(TM2), that hydrolyze 51 and 63 esters, respectively, while maintaining similar or higher turnover rates compared to the original enzyme. We hypothesized that the parameters identified here (the volume, size, exposure, enclosure, hydrophobicity, and hydrophilicity of the active site cavity and its tightness) can serve in the future to expand the substrate spectra of esterases and thus expand their use in biotechnology and synthetic chemistry.

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