Vazquez-Albacete Dario, Montefiori Marco, Kol Stefan, Motawia Mohammed Saddik, Møller Birger Lindberg, Olsen Lars, Nørholm Morten H H
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.
Faculty of Health and Medical Sciences, Department of Drug Design and Pharmacology, University of Copenhagen, Denmark.
Phytochemistry. 2017 Mar;135:8-17. doi: 10.1016/j.phytochem.2016.11.013. Epub 2017 Jan 11.
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.
植物细胞色素P450具有广泛的多样性和膜结合特性，这使得研究这类酶的结构特征具有挑战性，尤其是在准确的分子间酶-底物相互作用方面。为了解决这个问题，我们在此应用一种改进的混合结构策略对植物细胞色素P450进行同源建模。这使得基于蛋白质序列中的保守基序和二级结构预测来阐明结构成为可能。我们对研究充分的高粱细胞色素P450 CYP79A1进行了建模，该酶催化生氰糖苷蜀黍苷生物合成的第一步。对接实验确定了活性位点中参与底物结合并促进催化作用的关键区域。精氨酸152和苏氨酸534被确定为与底物相互作用的关键残基。该模型通过定点诱变进行了实验验证。新的CYP79A1模型为氰苷生物合成初始步骤的机制提供了详细见解。该方法可以指导其他膜结合细胞色素P450的功能表征，并为阐明其他植物细胞色素P450关键结构-功能关系提供结构指导。
