title: The CYP79A1 catalyzed conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime unravelled using an improved method for homology modeling.
abstract: The vast diversity and membrane-bound nature of plant P450s makes it challenging to study the structural characteristics of this class of enzymes especially with respect to accurate intermolecular enzyme-substrate interactions. To address this problem we here apply a modified hybrid structure strategy for homology modeling of plant P450s. This allows for structural elucidation based on conserved motifs in the protein sequence and secondary structure predictions. We modeled the well-studied Sorghum bicolor cytochrome P450 CYP79A1 catalyzing the first step in the biosynthesis of the cyanogenic glucoside dhurrin. Docking experiments identified key regions of the active site involved in binding of the substrate and facilitating catalysis. Arginine 152 and threonine 534 were identified as key residues interacting with the substrate. The model was validated experimentally using site-directed mutagenesis. The new CYP79A1 model provides detailed insights into the mechanism of the initial steps in cyanogenic glycoside biosynthesis. The approach could guide functional characterization of other membrane-bound P450s and provide structural guidelines for elucidation of key structure-function relationships of other plant P450s.
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.
authors.forename: Dario
authors.initials: D
authors.lastname: Vazquez-Albacete
authors.affiliation: Faculty of Health and Medical Sciences, Department of Drug Design and Pharmacology, University of Copenhagen, Denmark.
authors.forename: Marco
authors.initials: M
authors.lastname: Montefiori
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kol
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark; Center for Synthetic Biology bioSYNergy, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark.
authors.forename: Mohammed Saddik
authors.initials: MS
authors.lastname: Motawia
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark; Center for Synthetic Biology bioSYNergy, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark.
authors.forename: Birger Lindberg
authors.initials: BL
authors.lastname: Møller
authors.affiliation: Faculty of Health and Medical Sciences, Department of Drug Design and Pharmacology, University of Copenhagen, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Olsen
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark; Center for Synthetic Biology bioSYNergy, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark. Electronic address: morno@biosustain.dtu.dk.
authors.forename: Morten H H
authors.initials: MH
authors.lastname: Nørholm
mesh_terms: D001120:Arginine / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D010091:Oximes / Q000378:metabolism*; D045868:Sorghum / Q000201:enzymology*; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D010091:Oximes; C020363:4-hydroxyphenylacetaldoxime; D013912:Threonine; D014443:Tyrosine; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; C089817:cytochrome P450TYR
keywords: Cyanogenic glucosides; Cytochrome P450; Docking; Enzymes; Homology modeling; Membrane proteins