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Improvement of α-amino Ester Hydrolase Stability via Computational Protein Design

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Abstract

Amino ester hydrolases (AEHs) are capable of rapid synthesis of cephalexin but suffer from rapid deactivation even at low temperatures. Previous efforts to engineer AEH have generated several improved variants but have been limited in scope in part due to limitations in activity assay throughput for β-lactam synthesis reactions. Rational design of ‘whole variants’ was explored to rapidly improve AEH thermostability by mutating between 3–15% of residues. Most variants were found to be inactive due to a mutated calcium binding site, the function of which has not previously been described. Four active variants, all with improved melting temperatures, were characterized in terms of synthesis and hydrolysis activity, melting temperature, and deactivation at 25°C. Two variants were found to have improved total turnover numbers relative to the initial AEH variant; however, a clear tradeoff exists between improved stability and overall activity of each variant.

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Acknowledgements

This work was supported by the U.S. Food and Drug Administration Center for Drug Evaluation and Research through Grant U01FD006484, which is gratefully acknowledged. C.E.L. gratefully acknowledges funding by the U.S National Science Foundation through the Graduate Research Fellowship Program (GRFP) under Grant No. DGE-1650044.

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Authors and Affiliations

  1. Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, 30332, Georgia

    Colton E. Lagerman, Emily A. Joe, Martha A. Grover, Ronald W. Rousseau & Andreas S. Bommarius

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  1. Colton E. Lagerman
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  2. Emily A. Joe
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  3. Martha A. Grover
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  4. Ronald W. Rousseau
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  5. Andreas S. Bommarius
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Contributions

C.E.L., M.A.G., R.W.R., and A.S.B. developed the concept, C.E.L., E.J., M.A.G., R.W.R., and A.S.B. developed the methodology, C.E.L., E.J., M.A.G., R.W.R., and A.S.B. conducted the investigation, M.A.G., R.W.R., A.S.B. acquired the funding for the project, M.A.G., R.W.R., and A.S.B. administered the project, C.E.L., E.J., M.A.G., R.W.R., and A.S.B. wrote the original draft, and C.E.L., E.J., M.A.G., R.W.R., and A.S.B. reviewed and edited the manuscript.

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Correspondence to Andreas S. Bommarius.

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Lagerman, C.E., Joe, E.A., Grover, M.A. et al. Improvement of α-amino Ester Hydrolase Stability via Computational Protein Design. Protein J 42, 675–684 (2023). https://doi.org/10.1007/s10930-023-10155-z

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