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Expression, Purification, and Biochemical Characterization of the Flavocytochrome P450 CYP505A30 from Myceliophthora
George J Baker1, Hazel M Girvan1, Sarah Matthews1
1Centre for Synthetic Biology of Fine and Speciality Chemicals (SYNBIOCHEM), School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
Abstract:
The cytochrome P450/P450 reductase fusion enzyme CYP505A30 from the thermophilic fungus Myceliophthora thermophila and its heme (P450) domain were expressed in Escherichia coli and purified using affinity, ion exchange, and size exclusion chromatography. CYP505A30 binds straight chain fatty acids (from ∼C10 to C20), with highest affinity for tridecanoic acid (KD = 2.7 microM). Reduced nicotinamide adenine dinucleotide phosphate is the preferred reductant for CYP505A30 (KM = 3.1 microM compared to 330 microM for reduced nicotinamide adenine dinucleotide in cytochrome c reduction). Electron paramagnetic resonance confirmed cysteine thiolate coordination of heme iron in CYP505A30 and its heme domain. Redox potentiometry revealed an unusually positive midpoint potential for reduction of the flavin adenine dinucleotide and flavin mononucleotide cofactors (E0' ∼ -118 mV), and a large increase in the CYP505A30 heme domain FeIII/FeII redox couple (ca. 230 mV) on binding arachidonic acid substrate. This switch brings the ferric heme iron potential into the same range as that of the reductase flavins. Multiangle laser light scattering analysis revealed CYP505A30's ability to dimerize, whereas the heme domain is monomeric. These data suggest CYP505A30 may function catalytically as a dimer (as described for Bacillus megaterium P450 BM3), and that binding interactions between CYP505A30 heme domains are not required for dimer formation. CYP505A30 catalyzed hydroxylation of straight chain fatty acids at the ω-1 to ω-3 positions, with a strong preference for ω-1 over ω-3 hydroxylation in the oxidation of dodecanoic and tetradecanoic acids (88 vs 2% products and 63 vs 9% products, respectively). CYP505A30 has important structural and catalytic similarities to P450 BM3 but distinct regioselectivity of lipid substrate oxidation with potential biotechnological applications.
Insights
The thermophilic fungus CYP505A30 enzyme was purified and characterized, showing high affinity for fatty acids and catalyzing specific hydroxylation. This enzyme shares similarities with P450 BM3, indicating potential biotechnological applications.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Cytochrome P450 enzymes are crucial for diverse metabolic processes.
- Understanding the structure-function relationship of P450s, like CYP505A30 from *Myceliophthora thermophila*, is vital for biotechnological advancements.
- Fusion enzymes combining P450 and reductase domains offer unique catalytic properties.
Purpose of the Study:
- To express, purify, and biochemically characterize the CYP505A30 fusion enzyme and its heme domain from *Myceliophthora thermophila*.
- To investigate the substrate binding, redox properties, and catalytic activity of CYP505A30.
- To compare CYP505A30 with other known P450 enzymes, such as P450 BM3, for potential applications.
Main Methods:
- Expression in *Escherichia coli* and purification using chromatography techniques.
- Enzyme kinetics, including substrate binding affinity (KD) and reductase preference (KM).
- Spectroscopic methods (EPR, redox potentiometry) and multiangle laser light scattering for structural and redox analysis.
Main Results:
- CYP505A30 exhibits high affinity for straight-chain fatty acids, particularly tridecanoic acid.
- The enzyme prefers reduced nicotinamide adenine dinucleotide phosphate as a reductant and shows unique redox potentials.
- CYP505A30 catalyzes regioselective hydroxylation of fatty acids, primarily at the ω-1 position, and can dimerize catalytically.
Conclusions:
- CYP505A30 possesses distinct structural and catalytic features, including substrate specificity and dimerization, similar to P450 BM3.
- The enzyme's regioselectivity in lipid oxidation suggests potential for tailored biotechnological applications.
- Further research into CYP505A30 could lead to novel biocatalysts for industrial processes.
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