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Title: Structural and Mechanistic Analysis of Trichodiene Synthase Using Site-Directed Mutagenesis: Probing the Catalytic Function of Tryosine-295 and the Asparagine-225/Serine-229/Glutamate-233-Mg2+ B Motif

Abstract

Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the 'aspartate-rich' motif D100DXX(D/E) that coordinates to Mg{sup 2+}{sub A} and Mg{sup 2+}{sub C} source, and the 'NSE/DTE' motif N225DXXSXXXE that chelates Mg{sup 2+}{sub b} (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
959638
Report Number(s):
BNL-82624-2009-JA
Journal ID: ISSN 0003-9861; ABBIA4; TRN: US201016%%782
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Archives of Biochemistry and Biophysics
Additional Journal Information:
Journal Volume: 469; Journal Issue: 2; Journal ID: ISSN 0003-9861
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; CATALYSIS; CHELATES; CRYSTAL STRUCTURE; CYCLIZATION; DISTRIBUTION; FUSARIUM; KINETICS; MUTAGENESIS; MUTANTS; SPECIFICITY; national synchrotron light source

Citation Formats

Vedula, L, Jiang, J, Zakharian, T, Cane, D, and Christianson, D. Structural and Mechanistic Analysis of Trichodiene Synthase Using Site-Directed Mutagenesis: Probing the Catalytic Function of Tryosine-295 and the Asparagine-225/Serine-229/Glutamate-233-Mg2+ B Motif. United States: N. p., 2008. Web. doi:10.1016/j.abb.2007.10.015.
Vedula, L, Jiang, J, Zakharian, T, Cane, D, & Christianson, D. Structural and Mechanistic Analysis of Trichodiene Synthase Using Site-Directed Mutagenesis: Probing the Catalytic Function of Tryosine-295 and the Asparagine-225/Serine-229/Glutamate-233-Mg2+ B Motif. United States. https://doi.org/10.1016/j.abb.2007.10.015
Vedula, L, Jiang, J, Zakharian, T, Cane, D, and Christianson, D. 2008. "Structural and Mechanistic Analysis of Trichodiene Synthase Using Site-Directed Mutagenesis: Probing the Catalytic Function of Tryosine-295 and the Asparagine-225/Serine-229/Glutamate-233-Mg2+ B Motif". United States. https://doi.org/10.1016/j.abb.2007.10.015.
@article{osti_959638,
title = {Structural and Mechanistic Analysis of Trichodiene Synthase Using Site-Directed Mutagenesis: Probing the Catalytic Function of Tryosine-295 and the Asparagine-225/Serine-229/Glutamate-233-Mg2+ B Motif},
author = {Vedula, L and Jiang, J and Zakharian, T and Cane, D and Christianson, D},
abstractNote = {Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the 'aspartate-rich' motif D100DXX(D/E) that coordinates to Mg{sup 2+}{sub A} and Mg{sup 2+}{sub C} source, and the 'NSE/DTE' motif N225DXXSXXXE that chelates Mg{sup 2+}{sub b} (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.},
doi = {10.1016/j.abb.2007.10.015},
url = {https://www.osti.gov/biblio/959638}, journal = {Archives of Biochemistry and Biophysics},
issn = {0003-9861},
number = 2,
volume = 469,
place = {United States},
year = {Tue Jan 01 00:00:00 EST 2008},
month = {Tue Jan 01 00:00:00 EST 2008}
}