Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot

Rousseau, Olivier; Quaglia, Daniela; Alejaldre, Lorea; Ouadhi, Sara; Pelletier, Joelle N.

Abstract

Through the application of a region-focused saturation mutagenesis and randomization approach, protein engineering of the Cal-A enzyme was undertaken with the goal of conferring new triglyceride selectivity. Little is known about the mode of triglyceride binding to Cal-A. Engineering Cal-A thus requires a systemic approach. Targeted and randomized Cal-A libraries were created, recombined using the Golden Gate approach and screened to detect variants able to discriminate between long-chain (olive oil) and short-chain (tributyrin) triglyceride substrates using a high-throughput in vivo method to visualize hydrolytic activity. Discriminative variants were analyzed using an in-house script to identify predominant substitutions. This approach allowed identification of variants that exhibit strong discrimination for the hydrolysis of short-chain triglycerides and others that discriminate towards hydrolysis of long-chain triglycerides. A clear pattern emerged from the discriminative variants, identifying the 217–245 helix-loop-helix motif as being a hot-spot for triglyceride recognition. This was the consequence of introducing the entire mutational load in selected regions, without putting a strain on distal parts of the protein. Our results improve our understanding of the Cal-A lipase mode of action and selectivity. This holistic perspective to protein engineering, where parts of the gene are individually mutated and the impact evaluated in the context of the whole protein, can be applied to any protein scaffold.

Citation

Rousseau, O., Quaglia, D., Alejaldre, L., Ouadhi, S., & Pelletier, J. N. (2019). Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot. PLoS ONE, 14(1), Article e0210100. https://doi.org/10.1371/journal.pone.0210100

• Journal Article Type: Article
• Acceptance Date: Dec 17, 2018
• Online Publication Date: Jan 14, 2019
• Publication Date: Jan 14, 2019
• Deposit Date: Jun 18, 2020
• Publicly Available Date: Jun 18, 2020
• Journal: PLOS ONE
• Electronic ISSN: 1932-6203
• Publisher: Public Library of Science
• Peer Reviewed: Peer Reviewed
• Volume: 14
• Issue: 1
• Article Number: e0210100
• DOI: https://doi.org/10.1371/journal.pone.0210100
• Keywords: General Biochemistry, Genetics and Molecular Biology; General Agricultural and Biological Sciences; General Medicine
• Public URL: https://nottingham-repository.worktribe.com/output/4672089
• Publisher URL: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210100