Dataset Information

P450 redox enzymes in the white rot fungus Phanerochaete chrysosporium: gene transcription, heterologous expression, and activity analysis on the purified proteins.

ABSTRACT: 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.

SUBMITTER: Subramanian V

PROVIDER: S-EPMC3121562 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

P450 redox enzymes in the white rot fungus Phanerochaete chrysosporium: gene transcription, heterologous expression, and activity analysis on the purified proteins.

Subramanian Venkataramanan V Doddapaneni Harshavardhan H Syed Khajamohiddin K Yadav Jagjit S JS

Current microbiology 20100311 4

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 show ...[more]

PMID: 20221604

Similar Datasets

Project description:The activity of a self-sufficient cytochrome P450 enzyme, CYP505D6, from the lignin-degrading basidiomycete Phanerochaete chrysosporium was characterized. Recombinant CYP505D6 was produced in Escherichia coli and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C9-18 carbon chain lengths as the substrates. Hydroxylation occurred at the ?-1 to ?-6 positions of such substrates with C9-15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at the ?-1 to ?-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active-site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid were analyzed. Compared with wild-type CYP505D6, its V51Y variant generated few products hydroxylated at the ?-4 to ?-6 positions. The products generated by wild-type CYP102A1 were hydroxylated at the ?-1 to ?-4 positions, whereas its Y51V variant generated ?-1 to ?-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regiospecificity of fatty acid hydroxylation, at least that at the ?-4 to ?-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry.IMPORTANCE Phanerochaete chrysosporium is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in this study, P. chrysosporium CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C9-15) and fatty acids (C9-15) at the ?-1 to ?-7 or ?-1 to ?-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than those of the known fused proteins CYP102A1 and CYP505A1. The substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications.

Dataset Information

P450 redox enzymes in the white rot fungus Phanerochaete chrysosporium: gene transcription, heterologous expression, and activity analysis on the purified proteins.

Publications

P450 redox enzymes in the white rot fungus Phanerochaete chrysosporium: gene transcription, heterologous expression, and activity analysis on the purified proteins.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets