Document Detail


Two distinct proton donors at the active site of Escherichia coli 2,4-dienoyl-CoA reductase are responsible for the formation of different products.
MedLine Citation:
PMID:  18171025     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
NADPH-dependent 2,4-dienoyl-CoA reductase (DCR) is one of the auxiliary enzymes required for the beta-oxidation of unsaturated fatty acids. Mutants of Escherichia coli DCR were generated by site-directed mutagenesis to explore the molecular mechanism of this enzyme. The Tyr166Phe mutant, which was expected to be inactive due to the loss of its putative proton donor residue, exhibited 27% of the wild-type activity. However, the product of the reduction was 3-enoyl-CoA instead of 2-enoyl-CoA, the normal product. Glu164 seems to function as proton donor in the Tyr166Phe mutant, because the Tyr166Phe/ Glu164Gln double mutant was inactive whereas the Glu164Ala mutant exhibited low but significant activity. His252 is important for the efficient operation of Tyr166 because a His252Ala mutation by itself reduced the activity of DCR by 3 orders of magnitude, whereas the Tyr166Phe/His252Ala double mutation exhibited 4.4% of the wild-type activity. This data supports a mechanism that has Tyr166 with the assistance of His252 acting as proton donor in the wild-type enzyme to produce 2-enoyl-CoA, whereas Glu164 serves as the proton donor in the absence of Tyr166 to yield 3-enoyl-CoA. A Cys337Ala mutation, which resulted in the loss of most of the iron and acid-labile sulfur, decreased the reductase activity more than 1000-fold. This observation agrees with the proposed operation of an intramolecular electron transport chain that is essential for the effective catalysis of E. coli DCR.
Authors:
Xi Tu; Paul A Hubbard; Jung-Ja P Kim; Horst Schulz
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2008-01-03
Journal Detail:
Title:  Biochemistry     Volume:  47     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2008 Jan 
Date Detail:
Created Date:  2008-01-22     Completed Date:  2008-03-14     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1167-75     Citation Subset:  IM    
Affiliation:
Department of Chemistry, City College, and Graduate School of the City University of New York, New York, New York 10031, USA.
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MeSH Terms
Descriptor/Qualifier:
Binding Sites
Chromatography, High Pressure Liquid
Cloning, Molecular
Electron Transport Chain Complex Proteins / genetics,  metabolism
Escherichia coli / enzymology*,  genetics
Gene Expression
Kinetics
Models, Molecular
Mutation / genetics
NADPH Oxidase / genetics,  metabolism
Oxidoreductases Acting on CH-CH Group Donors / chemistry,  genetics,  metabolism*
Protein Structure, Tertiary
Protons*
Grant Support
ID/Acronym/Agency:
GM008168/GM/NIGMS NIH HHS; GM29076/GM/NIGMS NIH HHS; RR03060/RR/NCRR NIH HHS
Chemical
Reg. No./Substance:
0/Electron Transport Chain Complex Proteins; 0/Protons; EC 1.3.-/Oxidoreductases Acting on CH-CH Group Donors; EC 1.3.1.34/2,4-dienoyl-CoA reductase; EC 1.6.3.1/NADPH Oxidase

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


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