Functional expression and characterization of recombinant NADPH-P450 reductase from Malassezia globosa.
Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7- ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa.
Department of Biological Sciences and Center for Biotechnology Research at UBITA (CBRU), Konkuk University, Seoul, 143-701, Korea.
Hwayoun
H
Lee
Hyoung-Goo
HG
Park
Young-Ran
YR
Lim
Im-Soon
IS
Lee
Beom Joon
BJ
Kim
Cheul-Hun
CH
Seong
Young-Jin
YJ
Chun
Donghak
D
Kim
D000595:Amino Acid Sequence; D045304:Cytochromes c / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D008290:Malassezia / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis
D011994:Recombinant Proteins; D009580:Nitroblue Tetrazolium; D009249:NADP; D045304:Cytochromes c; D009251:NADPH-Ferrihemoprotein Reductase