Direct coupling analysis improves the identification of beneficial amino acid mutations for the functional thermostabilization of a delicate decarboxylase
Abstract
The optimization of enzyme properties for specific reaction conditions enables their tailored use in biotechnology. Predictions using established computer-based methods, however, remain challenging, especially regarding physical parameters such as thermostability without concurrent loss of activity. Employing established computational methods such as energy calculations using FoldX can lead to the identification of beneficial single amino acid substitutions for the thermostabilization of enzymes. However, these methods require a three-dimensional (3D)-structure of the enzyme. In contrast, coevolutionary analysis is a computational method, which is solely based on sequence data. To enable a comparison, we employed coevolutionary analysis together with structure-based approaches to identify mutations, which stabilize an enzyme while retaining its activity. As an example, we used the delicate dimeric, thiamine pyrophosphate dependent enzyme ketoisovalerate decarboxylase (Kivd) and experimentally determined its stability represented by a T50 value indicating the temperature where 50% of enzymatic activity remained after incubation for 10 min. Coevolutionary analysis suggested 12 beneficial mutations, which were not identified by previously established methods, out of which four mutations led to a functional Kivd with an increased T50 value of up to 3.9°C.
Acknowledgments
K.S.R., M.P. and M.M. acknowledge funding via the Helmholtz programme ‘BioInterfaces in Technology and Medicine’. J.E. and A.S. are supported by the Helmholtz Association Initiative and Networking Fund under project number ZT-I-0003. We thank Anke Dech for help with the protein purification.
References
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© 2019 Walter de Gruyter GmbH, Berlin/Boston
Artikel in diesem Heft
- Frontmatter
- Highlight: Young research groups in Germany – continued
- Exceptionally versatile – arginine in bacterial post-translational protein modifications
- The Gid-complex: an emerging player in the ubiquitin ligase league
- Emerging RNA-binding roles in the TRIM family of ubiquitin ligases
- Nanomedical detection and downstream analysis of circulating tumor cells in head and neck patients
- More than just a pressure relief valve: physiological roles of volume-regulated LRRC8 anion channels
- Regulating the development of pulmonary Group 2 innate lymphoid cells
- Comparison of lipidic carrier systems for integral membrane proteins – MsbA as case study
- Direct coupling analysis improves the identification of beneficial amino acid mutations for the functional thermostabilization of a delicate decarboxylase
Artikel in diesem Heft
- Frontmatter
- Highlight: Young research groups in Germany – continued
- Exceptionally versatile – arginine in bacterial post-translational protein modifications
- The Gid-complex: an emerging player in the ubiquitin ligase league
- Emerging RNA-binding roles in the TRIM family of ubiquitin ligases
- Nanomedical detection and downstream analysis of circulating tumor cells in head and neck patients
- More than just a pressure relief valve: physiological roles of volume-regulated LRRC8 anion channels
- Regulating the development of pulmonary Group 2 innate lymphoid cells
- Comparison of lipidic carrier systems for integral membrane proteins – MsbA as case study
- Direct coupling analysis improves the identification of beneficial amino acid mutations for the functional thermostabilization of a delicate decarboxylase