P450 redox enzymes in the white rot fungus Phanerochaete chrysosporium: gene transcription, heterologous expression, and activity analysis on the purified proteins.
With an aim to understand the cytochrome P450 enzyme system in the white rot fungus Phanerochaete chrysosporium, here we report molecular characterization of its P450 redox proteins including the primary P450 oxidoreductase (POR) and two alternate P450 redox proteins cytochrome b5 (cyt b5) and cytochrome b5 reductase (cyt b5r) in terms of transcriptional regulation and heterologous expression. The transcript abundance followed the order POR > cyt b5r > cyt b5. Interestingly, the three genes showed an overall higher expression in the defined carbon-limited cultures with low nitrogen (LN) or high nitrogen (HN) versus the carbon-rich malt extract (ME) cultures. cDNA cloning and analysis revealed the following deduced protein characteristics: cyt b5 (238 amino acids, 25.38 kDa) and cyt b5r (321 amino acids, 35.52 kDa). Phylogenetic analysis revealed that the cloned cyt b5 belongs to a novel class of fungal cyt b5-like proteins. The two proteins cyt b5 and cyt b5r were heterologously expressed in E. coli and purified using affinity-based purification in an active form. The POR was heterologously expressed in Saccharomyces cerevisiae and was also purified in active form as evidenced by its cytochrome c reduction activity. This is the first report on cloning, heterologous expression, and purification of the alternate redox proteins cyt b5 and cyt b5r in E. coli and on yeast expression of POR from this model white rot fungus.
Subramanian
Venkataramanan
V
Department of Environmental Health, University of Cincinnati, Cincinnati, OH 45267, USA.
Doddapaneni
Harshavardhan
H
Syed
Khajamohiddin
K
Yadav
Jagjit S
JS
D002244:Carbon; D003001:Cloning, Molecular; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015786:Cytochromes b5 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018076:DNA, Complementary; D004785:Environmental Pollutants / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D020440:Gene Duplication; D015870:Gene Expression; D016681:Genome, Fungal; D008031:Lignin / Q000378:metabolism; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009584:Nitrogen; D010084:Oxidation-Reduction; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014158:Transcription, Genetic
D003470:Culture Media; D018076:DNA, Complementary; D004785:Environmental Pollutants; D005656:Fungal Proteins; D011994:Recombinant Proteins; D002244:Carbon; D008031:Lignin; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D042966:Cytochrome-B(5) Reductase; D009251:NADPH-Ferrihemoprotein Reductase; D009584:Nitrogen