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
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.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 959638
- Report Number(s):
- BNL-82624-2009-JA; ABBIA4; TRN: US201016%%782
- Journal Information:
- Archives of Biochemistry and Biophysics, Vol. 469, Issue 2; ISSN 0003-9861
- Country of Publication:
- United States
- Language:
- English
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