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Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race

Tsou, Chung-Yau, Matsunaga, Shigeki, Okada, Shigeru
Journal of bioscience and bioengineering 2018 v.125 no.1 pp. 30-37
Botryococcus braunii, Escherichia coli, NADP (coenzyme), affinity chromatography, amino acid sequences, amino acids, complementary DNA, cytochrome P-450, cytochrome c, enzyme activity, enzymes, microalgae, molecular cloning, molecular weight, open reading frames, pH, squalene, temperature, triterpenoids
The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out. The isolated cDNA contained an open reading frame (1998 bp) that encoded for a protein with 665 amino acid residues with a predicted molecular weight of 71.46 kDa and a theoretical pI of 5.49. Analysis of the deduced amino acid sequence revealed the presence of conserved motifs, including FMN, FAD, and NADPH binding domains, which are typical of other CPRs and necessary for enzyme activity. By truncation of the N-terminal transmembrane anchor and addition of a 6× His-tag, BbCPR was heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified recombinant enzyme showed optimal reducing activity of cytochrome c at around a neutral pH at a temperature range of 30–37°C. For steady state kinetic parameters, the recombinant enzyme had a km for cytochrome c and NADPH of 11.7±1.6 and 9.4±1.4 μM, and a kcat for cytochrome c and NADPH of 2.78±0.09 and 3.66±0.11 μmol/min/mg protein, respectively. This is the first study to perform the functional characterization of a CPR from eukaryotic microalgae.