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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
2
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pubmed:dateCreated |
1999-6-15
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pubmed:abstractText |
Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and long-chain fatty acids at the positions omega-1, omega-2, and omega-3. A rapid and continuous spectrophotometric activity assay for cytochrome P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-oxycarboxylic acids and the chromophore p-nitrophenolate was developed. In contrast to the commonly used activity assays for this enzyme, relying on the consumption of oxygen or NADPH or the use of 14C-labeled carboxylic acids, the pNCA assay can even be used with crude extracts of the recombinant enzyme from lysed Escherichia coli cells. The kinetics of p-nitrophenolate formation are directly measured at a wavelength of 410 nm using a spectrophotometer or microtiter plate reader. Sensitivity of the assay is greatly enhanced if p-nitrophenoxydodecanoic or p-nitrophenoxypentadecanoic acid are used with the F87A mutant instead of the wild-type P450 BM-3 enzyme.
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
May
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pubmed:issn |
0003-2697
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pubmed:author |
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pubmed:copyrightInfo |
Copyright 1999 Academic Press.
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pubmed:issnType |
Print
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pubmed:day |
1
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pubmed:volume |
269
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
359-66
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pubmed:dateRevised |
2008-11-21
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pubmed:meshHeading |
pubmed-meshheading:10222011-Bacillus megaterium,
pubmed-meshheading:10222011-Bacterial Proteins,
pubmed-meshheading:10222011-Catalytic Domain,
pubmed-meshheading:10222011-Colorimetry,
pubmed-meshheading:10222011-Cytochrome P-450 Enzyme System,
pubmed-meshheading:10222011-Escherichia coli,
pubmed-meshheading:10222011-Fatty Acids,
pubmed-meshheading:10222011-Magnetic Resonance Spectroscopy,
pubmed-meshheading:10222011-Mixed Function Oxygenases,
pubmed-meshheading:10222011-Models, Molecular,
pubmed-meshheading:10222011-NADPH-Ferrihemoprotein Reductase,
pubmed-meshheading:10222011-Point Mutation,
pubmed-meshheading:10222011-Protein Conformation,
pubmed-meshheading:10222011-Protein Engineering,
pubmed-meshheading:10222011-Recombinant Proteins,
pubmed-meshheading:10222011-Sensitivity and Specificity,
pubmed-meshheading:10222011-Spectrophotometry,
pubmed-meshheading:10222011-Substrate Specificity
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pubmed:year |
1999
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pubmed:articleTitle |
A continuous spectrophotometric assay for P450 BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A.
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pubmed:affiliation |
Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, Stuttgart, 70569, Germany.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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