Abstract
An N-terminally modified form of the Arabidopsis NADPH-cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2',5'-ADP-agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 micromol min(-1) mg(-1) and the K(m) for cytochrome c reduction was 15 +/- 2 microM. The purified NADPH-cytochrome P450 reductase was able to support function of CYP79B2.
Copyright 2000 Academic Press.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Amino Acid Sequence
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Arabidopsis / chemistry*
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Base Sequence
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Chromatography, Affinity
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Chromatography, Agarose
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Cloning, Molecular
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Cytochrome P-450 Enzyme System / metabolism
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Cytochrome c Group / chemistry
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Mixed Function Oxygenases / metabolism
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Molecular Sequence Data
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NADH, NADPH Oxidoreductases / chemistry
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NADH, NADPH Oxidoreductases / isolation & purification*
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NADH, NADPH Oxidoreductases / metabolism
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NADPH-Ferrihemoprotein Reductase
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Oxidation-Reduction
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Sequence Analysis, DNA
Substances
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Cytochrome c Group
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Recombinant Proteins
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Cytochrome P-450 Enzyme System
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Mixed Function Oxygenases
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cytochrome P450TYR
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NADH, NADPH Oxidoreductases
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NADPH-Ferrihemoprotein Reductase