Purification and characterization of a cytosolic cytochrome P450 from yeast Trichosporon cutaneum.
A soluble cytochrome P450 from the yeast Trichosporon cutaneum was purified to homogeneity, using ammonium sulfate fractionation followed by fast protein liquid chromatography (FPLC) with DEAE-cellulose and phenyl-Sepharose columns. This procedure resulted in a 45-fold increase in specific activity with an activity yield of 6.8%. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified enzyme was homogeneous and had a molecular mass of 45 kDa. The purified enzyme contained a heme group and had a characteristic absorption peak at 448 nm in the reduced carbon monoxide difference spectrum. This enzyme was a monomeric protein and catalyzed the conversion of salicylic acid to catechol in the presence of NADH or NADPH. The N-terminal amino acid sequence indicated that the Trichosporon cutaneum cytochrome P450 did not show homology to most eukaryotic cytochromes P450, but had a high degree of homology to one cytochrome P450, the nitric oxide reductase, of Fusarium oxysporum.
Yang
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Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079, USA.
Zhang
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Cerniglia
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D000595:Amino Acid Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification*; D003600:Cytosol / Q000201:enzymology; D008969:Molecular Sequence Data; D012459:Salicylates / Q000378:metabolism; D020156:Salicylic Acid; D014250:Trichosporon / Q000201:enzymology*
D012459:Salicylates; D003577:Cytochrome P-450 Enzyme System; D020156:Salicylic Acid