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Biochemical Characterization of the Cytochrome P450 CYP107CB2 from Bacillus lehensis G1

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Abstract

The bioconversion of vitamin D3 catalyzed by cytochrome P450 (CYP) requires 25-hydroxylation and subsequent 1α-hydroxylation to produce the hormonal activated 1α,25-dihydroxyvitamin D3. Vitamin D3 25-hydroxylase catalyses the first step in the vitamin D3 biosynthetic pathway, essential in the de novo activation of vitamin D3. A CYP known as CYP107CB2 has been identified as a novel vitamin D hydroxylase in Bacillus lehensis G1. In order to deepen the understanding of this bacterial origin CYP107CB2, its detailed biological functions as well as biochemical characteristics were defined. CYP107CB2 was characterized through the absorption spectral analysis and accordingly, the enzyme was assayed for vitamin D3 hydroxylation activity. CYP-ligand characterization and catalysis optimization were conducted to increase the turnover of hydroxylated products in an NADPH-regenerating system. Results revealed that the over-expressed CYP107CB2 protein was dominantly cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS–PAGE, indicative of CYP107CB2. Spectral analysis indicated that CYP107CB2 protein was properly folded and it was in the active form to catalyze vitamin D3 reaction at C25. HPLC and MS analysis from a reconstituted enzymatic reaction confirmed the hydroxylated products were 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3 when the substrates vitamin D3 and 1α-hydroxyvitamin D3 were used. Biochemical characterization shows that CYP107CB2 performed hydroxylation activity at 25 °C in pH 8 and successfully increased the production of 1α,25-dihydroxyvitamin D3 up to four fold. These findings show that CYP107CB2 has a biologically relevant vitamin D3 25-hydroxylase activity and further suggest the contribution of CYP family to the metabolism of vitamin D3.

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Acknowledgements

This work was financially supported by Research University Grant (05-02-12-2185RU) from Universiti Putra Malaysia. We acknowledge Malaysia Genome Institute (MGI) for providing the bacterial strain and its genome data. Swi See Ang would like to thank Ministry of Education, Malaysia for MyPhD scholarship award.

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Authors and Affiliations

  1. Enzyme and Microbial Technology Research Center, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia UPM, 43400, Serdang, Selangor, Malaysia

    Swi See Ang, Abu Bakar Salleh, Leow Thean Chor, Yahaya M. Normi, Bimo Ario Tejo & Mohd Basyaruddin Abdul Rahman

  2. Laboratory of Enzyme Technology, Institute of Bioscience, Universiti Putra Malaysia UPM, 43400, Serdang, Selangor, Malaysia

    Swi See Ang, Abu Bakar Salleh & Leow Thean Chor

  3. Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia UPM, 43400, Serdang, Selangor, Malaysia

    Abu Bakar Salleh

  4. Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia UPM, 43400, Serdang, Selangor, Malaysia

    Leow Thean Chor & Yahaya M. Normi

  5. Department of Chemistry, Faculty of Science, Universiti Putra Malaysia UPM, 43400, Serdang, Selangor, Malaysia

    Bimo Ario Tejo & Mohd Basyaruddin Abdul Rahman

  6. Faculty of Health and Life Sciences, Management and Science University, 40100, Shah Alam, Selangor, Malaysia

    Mariam-Aisha Fatima

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  1. Swi See Ang
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Contributions

SSA designed, performed the experiments, analyzed the data and prepared the manuscript. ALTC, BAT and MBAR involved in data analysis. YMN and MAF participated in research design, data interpretation and drafted the manuscript. ABS supervised the study, designed the experiment and revised the manuscript.

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Correspondence to Abu Bakar Salleh.

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Ang, S.S., Salleh, A.B., Chor, L.T. et al. Biochemical Characterization of the Cytochrome P450 CYP107CB2 from Bacillus lehensis G1. Protein J 37, 180–193 (2018). https://doi.org/10.1007/s10930-018-9764-z

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