Document Detail


Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis.
MedLine Citation:
PMID:  15978541     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.
Authors:
David C Hyatt; Rodney Croteau
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Archives of biochemistry and biophysics     Volume:  439     ISSN:  0003-9861     ISO Abbreviation:  Arch. Biochem. Biophys.     Publication Date:  2005 Jul 
Date Detail:
Created Date:  2005-07-04     Completed Date:  2005-09-01     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0372430     Medline TA:  Arch Biochem Biophys     Country:  United States    
Other Details:
Languages:  eng     Pagination:  222-33     Citation Subset:  IM    
Affiliation:
Institute of Biological Chemistry, Washington State University, Pullman, 99163-6340, USA.
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MeSH Terms
Descriptor/Qualifier:
Abies / enzymology*
Amino Acid Sequence
Amino Acid Substitution
Catalysis
DNA Mutational Analysis
Intramolecular Lyases / chemistry,  genetics*,  metabolism*
Molecular Sequence Data
Mutagenesis, Site-Directed
Sequence Homology, Amino Acid
Grant Support
ID/Acronym/Agency:
GM-31354/GM/NIGMS NIH HHS; R37 CA055254-14/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
EC 5.5.-/Intramolecular Lyases; EC 5.5.-/pinene cyclase I

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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