Expression, Purification, and Biochemical Characterization of the Flavocytochrome P450 CYP505A30 from Myceliophthora thermophila.

ACS Omega
George J BakerA W Munro

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 μM). Reduced nicotinamide adenine dinucleotide phosphate is the preferred reductant for CYP505A30 (KM = 3.1 μM compared to 330 μM 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 CYP505A3...Continue Reading

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Citations

Sep 30, 2018·Applied and Environmental Microbiology·Efstratios NikolaivitsEvangelos Topakas
Sep 5, 2019·Biotechnology and Bioengineering·Lisa Marie SchmitzStephan Lütz
Dec 18, 2020·Organic & Biomolecular Chemistry·Ana C EbrechtDiederik J Opperman
Nov 25, 2020·Chembiochem : a European Journal of Chemical Biology·Chun-Chi ChenRey-Ting Guo
Jul 11, 2021·Trends in Biotechnology·Soo-Jin YeomChul-Ho Yun

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Methods Mentioned

BETA
electrophoresis
Light Scattering
equilibrium binding
ion exchange chromatography
size exclusion chromatography
PCR
gel filtration

Software Mentioned

Clustal Omega
MALLS
GenScript
Origin
BLAST
FigTree
OriginLab
antiSMASH

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