Abstract
The human genome includes four cytochrome P450 2C subfamily enzymes, and CYP2C8 has generated research interest because it is subject to drug–drug interactions and various polymorphic outcomes. To address the structure-functional complexity of CYP2C8, its catalytic activity was studied using a directed evolution analysis. Consecutive rounds of random mutagenesis and screening using 6-methoxy-luciferin produced two mutants, which displayed highly increased luciferase activity. Wild-type and selected mutants were expressed on a large scale and purified. The expression levels of the D349Y and D349Y/V237A mutants were ~310 and 460 nmol per liter of culture, respectively. The steady-state kinetic analysis of paclitaxel 6α-hydroxylation showed that the mutants exhibited a 5–7-fold increase in kcat values and a 3–5-fold increase in catalytic efficiencies (kcat/KM). In arachidonic acid epoxidation, two mutants exhibited a 30–150-fold increase in kcat values and a 40–110-fold increase in catalytic efficiencies. The binding titration analyses of paclitaxel and arachidonic acid showed that the V237A mutation had a lower Kd value, indicating a tighter substrate-binding affinity. The structural analysis of CYP2C8 indicated that the D349Y mutation was close enough to the putative binding domain of the redox partner; the increase in catalytic activity could be partially attributed to the enhancement of the P450 coupling efficiency or electron transfer.
Highlights
Cytochrome P450 enzymes (P450, CYP) consist of a superfamily of heme-thiolate proteins and exhibit a unique spectral absorbance at 450 nm when their reduced forms are combined with carbon monoxide [1,2]
CYP2C8 participates in the conversion of arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which are involved in the regulation of physiological processes, including blood pressure regulation, platelet aggregation, vascular function, and pancreatic peptide hormone secretion [9,10,11]
Luciferin-ME was converted to luciferin by CYP2C8, and luminescence emission was
Summary
Cytochrome P450 enzymes (P450, CYP) consist of a superfamily of heme-thiolate proteins and exhibit a unique spectral absorbance at 450 nm when their reduced forms are combined with carbon monoxide [1,2]. The human genome contains 57 P450 genes, which are distributed in most autosomal chromosomes [3]. Among the many P450s, 12 enzymes from the P450 1, 2, and 3 families play central roles in the oxidative metabolism of xenobiotic chemicals, including most clinical drugs [4]. There are four genes of the cytochrome P450 2C subfamily of enzymes in the human genome, which are located in chromosome 10q24 in the centromere-2C18-2C19-2C9-2C8telomere order [5]. 6α-hydroxypaclitaxel, is a typical biomarker reaction of CYP2C8 [8]. CYP2C8 participates in the conversion of arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which are involved in the regulation of physiological processes, including blood pressure regulation, platelet aggregation, vascular function, and pancreatic peptide hormone secretion [9,10,11]. 18 allelic variants of CYP2C8 have been identified, and
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