Active-Site Mutagenesis of Fatty Acid Photodecarboxylase

Experimental and Computational Insight into Substrate Chain-Length Specificity

verfasst von: Santiago Nahuel Chanquia, Jan Philipp Bittner, Paul Santner, László Krisztián Szabó, Jakob Schelde Madsen, Marcus Lyngdahl Øhlenschlæger, Ahmad Gheis Sarvari, Aske Ho̷j Merrild, Kathrine Gravlund Fo̷nss, Daily Jaron, Linnea Lutz, Selin Kara, Bekir Engin Eser
Abstract: 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.
Organisationseinheit(en): Institut für Technische Chemie
Externe Organisation(en): Aarhus University
Technische Universität Hamburg (TUHH)
Typ: Artikel
Journal: ACS catalysis
Band: 14
Seiten: 15837-15849
Anzahl der Seiten: 13
ISSN: 2155-5435
Publikationsdatum: 01.11.2024
Publikationsstatus: Veröffentlicht
Peer-reviewed: Ja
ASJC Scopus Sachgebiete: Katalyse, Allgemeine Chemie
Elektronische Version(en): https://doi.org/10.1021/acscatal.4c02970 (Zugang: Geschlossen)

BibTeX

@article{937c920829b641e28b08d9a2af1238e3,
title = "Active-Site Mutagenesis of Fatty Acid Photodecarboxylase: Experimental and Computational Insight into Substrate Chain-Length Specificity",
abstract = "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{\textquoteright}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.",
keywords = "biocatalysis, drop-in biofuel, fatty acid photodecarboxylase, molecular dynamics simulations, photoenzyme, protein engineering, substrate specificity",
author = "Chanquia, {Santiago Nahuel} and Bittner, {Jan Philipp} and Paul Santner and Szab{\'o}, {L{\'a}szl{\'o} Kriszti{\'a}n} and Madsen, {Jakob Schelde} and {\O}hlenschl{\ae}ger, {Marcus Lyngdahl} and Sarvari, {Ahmad Gheis} and Merrild, {Aske Ho̷j} and Fo̷nss, {Kathrine Gravlund} and Daily Jaron and Linnea Lutz and Selin Kara and Eser, {Bekir Engin}",
note = "Publisher Copyright: {\textcopyright} 2024 American Chemical Society.",
year = "2024",
month = nov,
day = "1",
doi = "10.1021/acscatal.4c02970",
language = "English",
volume = "14",
pages = "15837--15849",
journal = "ACS catalysis",
issn = "2155-5435",
publisher = "American Chemical Society",
number = "21",
}