Biomol Ther.  2023 Jan;31(1):82-88. 10.4062/biomolther.2022.084.

Functional Characterization of Drosophila melanogaster CYP6A8 Fatty Acid Hydroxylase

• Lee SA
• Kim V
• Choi B
• Lee H
• Chun YJ
• Cho KS
• Kim D

• KMID: 2537584
• DOI: http://doi.org/10.4062/biomolther.2022.084

Abstract

Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. Here, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni²⁺ -nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type І spectral changes, with K_d values 28 ± 4 and 144 ± 20 μM, respectively. Ultra-performance liquid chromatography–mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a k_cat value of 0.038 ± 0.002 min –1 and a K_m value of 10 ± 2 μM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a k_cat value of 0.135 ± 0.007 min –1 and a K_m value of 21 ± 4 μM. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.

Keyword

P450; CYP6A8; Drosophila melanogaster; Lauric acid; Capric acid; Mass spectrometry