TY - JOUR

T1 - Active-Site Mutagenesis of Fatty Acid Photodecarboxylase

T2 - Experimental and Computational Insight into Substrate Chain-Length Specificity

AU - Chanquia, Santiago Nahuel

AU - Bittner, Jan Philipp

AU - Santner, Paul

AU - Szabó, László Krisztián

AU - Madsen, Jakob Schelde

AU - Øhlenschlæger, Marcus Lyngdahl

AU - Sarvari, Ahmad Gheis

AU - Merrild, Aske Ho̷j

AU - Fo̷nss, Kathrine Gravlund

AU - Jaron, Daily

AU - Lutz, Linnea

AU - Kara, Selin

AU - Eser, Bekir Engin

N1 - Publisher Copyright: © 2024 American Chemical Society.

PY - 2024/11/1

Y1 - 2024/11/1

N2 - 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.

AB - 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.

KW - biocatalysis

KW - drop-in biofuel

KW - fatty acid photodecarboxylase

KW - molecular dynamics simulations

KW - photoenzyme

KW - protein engineering

KW - substrate specificity

UR - http://www.scopus.com/inward/record.url?scp=85206818588&partnerID=8YFLogxK

U2 - 10.1021/acscatal.4c02970

DO - 10.1021/acscatal.4c02970

M3 - Article

AN - SCOPUS:85206818588

VL - 14

SP - 15837

EP - 15849

JO - ACS catalysis

JF - ACS catalysis

SN - 2155-5435

IS - 21

ER -