Recombinant Candida tropicalis NADPH--cytochrome P450 reductase (NCP1)
Description
Overview and Gene Context
NCP1 is a full-length protein (1–680 amino acids) encoded by the NCP1 gene in C. tropicalis. It belongs to the cytochrome P450 reductase (CPR) family, which includes homologs such as CPR-a and CPR-b in C. tropicalis . The enzyme contains FAD and FMN cofactors, enabling bidirectional electron transfer .
Key Features:
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Genomic Context: NCP1 is part of the C. tropicalis genome, with allelic variants (CPR-a/b) showing high sequence homology .
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Industrial Relevance: NCP1 supports ω-oxidation pathways, crucial for dicarboxylic acid production in C. tropicalis .
Structure and Biochemical Properties
The recombinant NCP1 protein is produced in E. coli with an N-terminal His-tag for purification . Structural and functional studies reveal:
Functional Insights:
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Electron Transfer: NCP1 transfers electrons from NADPH to FAD → FMN → CYP enzymes, enabling oxygen activation for hydroxylation or oxidation .
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Uncoupling and ROS Production: C. tropicalis CPR (CtCPR) exhibits 30% uncoupling during cytochrome c reduction, generating reactive oxygen species (ROS) independent of reaction conditions .
Heterologous Expression and Validation
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Expression in E. coli: Recombinant NCP1 retains functional activity, as demonstrated by cytochrome c reduction assays .
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Antibody Development: A specific antibody targeting the C-terminal peptide (SEQ ID NO:34) detects NCP1 in Western blots, confirming expression levels in engineered C. tropicalis strains .
Role in P450 Systems
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CYP Coupling: NCP1 supports P450-mediated reactions, such as 7-ethoxyresorufin O-deethylation when paired with human P450 1A2 .
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Industrial Applications: Overexpression of NCP1 under the POX4 promoter enhances reductase activity in C. tropicalis, boosting dicarboxylic acid yield .
Biotechnological Uses
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Dicarboxylic Acid Production: NCP1 enhances ω-oxidation in C. tropicalis, critical for converting fatty acids to industrial dicarboxylic acids .
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Enzyme Engineering: Recombinant NCP1 serves as a model for studying CPR-CYP interactions and uncoupling mechanisms .
Research Tools
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Antibody-Based Detection: The NCP1-specific antibody enables precise quantification in engineered strains .
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Functional Assays: NCP1 is used to reconstitute P450 activity in vitro, aiding studies on fungal metabolism and detoxification .
Data Tables
Table 2: Key Research Advances in NCP1
Product Specs
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Target Background
This enzyme facilitates electron transfer from NADPH to cytochrome P450 in microsomes. It also mediates electron transfer to heme oxygenase and cytochrome b5 and is involved in ergosterol biosynthesis.
