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Structure-based Mutational Studies of D-3-hydroxybutyrate Dehydrogenase for Substrate Recognition of Aliphatic Hydroxy Acids with a Variable Length of Carbon Chain

  • Research Paper
  • Protein Engineering and Enzyme Biotechnology
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Abstract

Native 3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis can catalyze the reversible reduction of acetoacetate, a four carbon chain oxo acid. This enzyme has been engineered to enable the reduction of levulinic acid, with one carbon longer than acetoacetate. In this study, the native and engineered enzymes were subjected to the catalysis of oxo acids with a carbon chain length of 3 to 8, in order to examine the capability of the enzyme to work on various platform chemicals. The engineered enzyme could reduce the C7 and C8 oxo acids whereas the wild-type had no activity on these substrates. Docking simulation has indicated Tyr155 and Ser142 are key residues for the catalysis. In addition, stable hydrogen bond formation between Gln196 and the substrates affects the turnover rate. Mutation sites in the engineered enzyme were focused on creating larger active site volume for substrates with extended chain lengths. Both qualitative and quantitative structural basis for the enzyme substrate specificity on alpha, beta, gamma and omega hydroxy acids could be elucidated.

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Acknowledgements

This research was supported by the C1 Gas Refinery Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2015M3D3A1A01064929).

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Authors and Affiliations

  1. Program of Bioengineering, Seoul National University, Seoul, 08826, Korea

    Hoe-Suk Lee

  2. Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, 04107, Korea

    Jeong Geol Na & Jinwon Lee

  3. Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneung, 25457, Korea

    Young Joo Yeon

  4. C1 Gas Refinery R&D Center, Sogang University, Seoul, 04107, Korea

    Hoe-Suk Lee, Jeong Geol Na, Jinwon Lee & Young Joo Yeon

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  1. Hoe-Suk Lee
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Correspondence to Jinwon Lee or Young Joo Yeon.

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Lee, HS., Na, J.G., Lee, J. et al. Structure-based Mutational Studies of D-3-hydroxybutyrate Dehydrogenase for Substrate Recognition of Aliphatic Hydroxy Acids with a Variable Length of Carbon Chain. Biotechnol Bioproc E 24, 605–612 (2019). https://doi.org/10.1007/s12257-019-0135-1

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  • DOI: https://doi.org/10.1007/s12257-019-0135-1

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