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Active-site mutagenesis of fatty acid photodecarboxylase: experimental and computational insight into substrate chain-length specificity
Publikationstyp
Journal Article
Date Issued
2024-10-10
Sprache
English
Author(s)
Szabó, László Krisztián
Madsen, Jakob Schelde
Øhlenschlæger, Marcus Lyngdahl
Sarvari, Ahmad Gheis
Merrild, Aske Ho̷j
Fo̷nss, Kathrine Gravlund
Jaron, Daily
Lutz, Linnea Ute
Journal
Volume
14
Issue
21
Start Page
15837
End Page
15849
Citation
ACS Catalysis 14 (21): 15837-15849 (2024)
Publisher DOI
Scopus ID
Publisher
ACS
Fatty acid photodecarboxylase (FAP), a microalgal enzyme, is one of the rare photoenzymes found in nature. Since its discovery in 2017, FAP has made a huge impact in the field of photobiocatalysis, being so far the only photoenzyme with potential applicability for organic synthesis. Furthermore, among all studied enzymes to date, FAP is one of the most promising candidates for in vitro feasible biofuel production from oil. One field of study for FAP has been broadening its substrate scope and modulating substrate selectivity. In order to get insight into the enzyme’s substrate selectivity, as well as to generate a toolbox of mutant enzymes with distinct substrate preferences toward medium- and long-chain fatty acids, in this work, we carried out extensive mutagenesis of the active-site residues of FAP from Chlorella variabilis (CvFAP). Particularly, we performed partial-site saturation mutagenesis for the Y466 position due to its key location at the active site. Our experimental and computational analysis indicated a correlation between the exchanged amino acid type and the observed activity, demonstrating that the conventional binding mode of long-chain fatty acids is destabilized by charged amino acid residues, leading to a nonproductive binding conformation characterized by a compact folded form. Mutagenesis of other key residues around the substrate binding site led to variants with selectivity toward medium-chain or long-chain fatty acids. For example, we obtained enzyme variants that are highly selective toward either C12:0, C14:0, or C18:0/C18:1 fatty acids. Selectivity patterns agreed very well with the distances between the FAD cofactor and substrate, as calculated by our molecular dynamics simulations. Furthermore, we report unexplored activity of the wild-type CvFAP toward C20:1 and C22:1 fatty acids, which are major components of jojoba oil and rapeseed oil, respectively.
Subjects
biocatalysis
drop-in biofuel
fatty acid photodecarboxylase
molecular dynamics simulations
photoenzyme
protein engineering
substrate specificity
DDC Class
500: Science