Study of 2,4-dienoyl-CoA reductases from Escherichia coli and rat.
Item
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Title
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Study of 2,4-dienoyl-CoA reductases from Escherichia coli and rat.
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Identifier
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AAI3284383
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identifier
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3284383
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Creator
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Tu, Xi.
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Contributor
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Adviser: Horst Schulz
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Date
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2007
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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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 E. 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 three 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.;It has been reported that a second DCR is present in rat mitochondria, which so far has not been purified or characterized nor has its cDNA been cloned (Hakkola & Hiltunen (1993) Eur. J. Biochem. 215, 199-204). As a first step toward confirming the existence of this enzyme and characterizing it, a soluble extract of rat liver was fractionated on hydroxyapatite and assayed for DCR activity. Three peaks of DCR activity were detected. The DCRs corresponding to the very small first peak and the large third peak were identified by immunoblotting as peroxisomal DCR and the well known mitochondrial DCR, respectively. The DCR that eluted at the intermediate position was immunologically unrelated to the two known mammalian DCRs but was detected in an extract from rat liver mitochondria. It is concluded that rat mitochondria and most likely other mammalian mitochondria contain a second DCR distinct from the known mammalian DCRs.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
