Poster: Enzymology

Abs # 244: Structural Biology Study of Isoflavonoid Phytoalexin Biosynthesis

Presenter:	Wang, Xiaoqiang , xwang@noble.org
Authors	Wang, Xiaoqiang  (A)   He, Xian-Zhi  (A)   Liu, Sijiu  (A)   Lin, Jianqiao  (A)   Blount, Jack W. (A)   Deavours, Bettina E. (A)   Dixon, Richard A. (A)
Affiliations:	(A): The Samuel Roberts Noble Foundation

Isoflavonoids are very valuable secondary metabolites primarily produced in leguminous plants. They have significant health benefit for humans. As phytoalexins, they also play important roles in plant defense against microorganisms. Medicarpin, the major isoflavonoid phytoalexin in alfalfa responding to fungal pathogen attack, is synthesized via the isoflavonoid branch of phenylpropanoid metabolism. There are several enzymes involved in this biosynthetic process. Isoflavone synthase (IFS, or 2-HIS (2-hydroxy-isoflavanone synthase)) is a key player that catalyses the entry-point reaction into isoflavonoid phytoalexin biosynthesis. Isoflavone reductase (IFR) and vestitone reductase (VR) are two enzymes involved in the latter part of the pathway. To improve our understanding of this biosynthetic pathway in detail, we are investigating the three dimensional structures of these enzymes, the interactions with their substrates/co-factors, and their catalytic mechanisms. IFS is a membrane associated cytochrome P450 enzyme. We designed several constructs to remove the N-terminal membrane binding achor and cloned them into several E. coli vectors to identify a high expression system that allows us to get sufficient of protein for structural studies. IFR and VR have been cloned into pET28a and pET15b vectors, respectively, and highly expressed in E. coli. These two proteins were purified by column chromatography. Crystallization trials are in progress.