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Title: Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase

Abstract

To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the first molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 {angstrom} of each other provides the basis for enhanced in vivo synthesis of resveratrol.

Authors:
; ; ; ;  [1];  [2]
  1. (WU)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
NSFDOE - OTHER
OSTI Identifier:
1046239
Resource Type:
Journal Article
Journal Name:
J. Am. Chem. Soc.
Additional Journal Information:
Journal Volume: 133; Journal Issue: (51) ; 12, 2011; Journal ID: ISSN 0002-7863
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; ARABIDOPSIS; BIOSYNTHESIS; CRYSTAL STRUCTURE; EFFICIENCY; ENZYME ACTIVITY; ENZYMES; GRAPES; IN VIVO; KINETICS; LIGASES; PROTEINS; STILBENE; SYNTHESIS; YEASTS

Citation Formats

Wang, Yechun, Yi, Hankuil, Wang, Melissa, Yu, Oliver, Jez, Joseph M., and Danforth). Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase. United States: N. p., 2012. Web. doi:10.1021/ja2085993.
Wang, Yechun, Yi, Hankuil, Wang, Melissa, Yu, Oliver, Jez, Joseph M., & Danforth). Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase. United States. doi:10.1021/ja2085993.
Wang, Yechun, Yi, Hankuil, Wang, Melissa, Yu, Oliver, Jez, Joseph M., and Danforth). Wed . "Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase". United States. doi:10.1021/ja2085993.
@article{osti_1046239,
title = {Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase},
author = {Wang, Yechun and Yi, Hankuil and Wang, Melissa and Yu, Oliver and Jez, Joseph M. and Danforth)},
abstractNote = {To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the first molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 {angstrom} of each other provides the basis for enhanced in vivo synthesis of resveratrol.},
doi = {10.1021/ja2085993},
journal = {J. Am. Chem. Soc.},
issn = {0002-7863},
number = (51) ; 12, 2011,
volume = 133,
place = {United States},
year = {2012},
month = {10}
}