Candida albicans NADPH-P450 reductase: expression, purification, and characterization of recombinant protein.
Candida albicans is responsible for serious fungal infections in humans. Analysis of its genome identified NCP1 gene coding for a putative NADPH-P450 reductase (NPR) enzyme. This enzyme appears to supply reducing equivalents to cytochrome P450 or heme oxygenase enzymes for fungal survival and virulence. In this study, we report the characterization of the functional features of NADPH-P450 reductase from C. albicans. The recombinant C. albicans NPR protein harboring a 6x(His)-tag was expressed heterologously in Escherichia coli, and was purified. Purified C. albicans NPR has an absorption maximum at 453 nm, indicating the feature of an oxidized flavin cofactor, which was decreased by the addition of NADPH. It also evidenced NADPH-dependent cytochrome c or nitroblue tetrazolium reducing activity. This purified reductase protein was successfully able to substitute for purified mammalian NPR in the reconstitution of the human P450 1A2-catalyzed O-deethylation of 7-ethoxyresorufin. These results indicate that purified C. albicans NPR is an orthologous reductase protein that supports cytochrome P450 or heme oxygenase enzymes in C. albicans.
Park
Hyoung-Goo
HG
Department of Biological Sciences, Konkuk University, Seoul 143-701, Republic of Korea.
Lim
Young-Ran
YR
Eun
Chang-Yong
CY
Han
Songhee
S
Han
Jung-Soo
JS
Cho
Kyoung Sang
KS
Chun
Young-Jin
YJ
Kim
Donghak
D
D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology*; D002384:Catalysis; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013053:Spectrophotometry
D011994:Recombinant Proteins; D009249:NADP; D009251:NADPH-Ferrihemoprotein Reductase