Purification and characterization of cytosolic cytochrome P450 forms from yeasts belonging to the genus Trichosporon.
The yeast Trichosporon spec. SBUG 752 isolated from soil produced cytochrome P450 during the stationary phase of growth on glucose. After cell disruption and ultracentrifugation, large amounts of P450 (250 pmol/mg protein) were found in the cytosolic fraction. In contrast, no P450 was detectable in the microsomes. Similar results were also obtained from some other yeast species of the genus Trichosporon. After purification to electrophoretic homogeneity, the P450 from Trichosporon spec. SBUG 752 migrated in SDS-PAGE with an apparent Mr of 43,000. Final purification by isoelectric focusing yielded two different isoenzymes in their spectrally active state-P450TS1 and P450TS2-having pI values of 5.9 and 6.2, respectively. Partial N-terminal amino acid sequencing revealed a high degree of sequence homology between P450TS1 and P450TS2 and their close relationship to the soluble P450 forms of the CYP55 family which are known to act as nitric oxide reductases in some filamentous fungi. The P450TS1 from Trichosporon spec. SBUG 752 catalyzed nitric oxide reduction under anaerobic conditions in an NADH- and NADPH-dependent manner-an activity not yet described for yeasts. These results demonstrate the existence of soluble P450 forms in yeasts exhibiting functional and structural characteristics similar to those of the P450 forms of the CYP55 family.
Stündl
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Institute of Microbiology and Molecular Biology, University of Greifswald, 17487 Greifswald, Jahnstrasse 15, Germany. stuendl@microbio1.biologie.uni-greifswald.de
Schmidt
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Scheller
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Schmid
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Schunck
W H
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Schauer
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D000595:Amino Acid Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification*; D003600:Cytosol / Q000201:enzymology*; D007424:Intracellular Fluid / Q000201:enzymology; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013053:Spectrophotometry; D014250:Trichosporon / Q000201:enzymology* / Q000254:growth & development
D010446:Peptide Fragments; D009569:Nitric Oxide; D003577:Cytochrome P-450 Enzyme System