Biochemical Characterization of the Cytochrome P450 CYP107CB2 from Bacillus lehensis G1

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 nov...

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Bibliographic Details
Published in:The Protein Journal 2018-04, Vol.37 (2), p.180-193
Main Authors: Ang, Swi See, Salleh, Abu Bakar, Chor, Leow Thean, Normi, Yahaya M., Tejo, Bimo Ario, Rahman, Mohd Basyaruddin Abdul, Fatima, Mariam-Aisha
Format: Article
Language:English
Subjects:
Alfacalcidol
Analysis
Animal Anatomy
Bacillus lehensis
Biochemical characteristics
Biochemistry
Bioconversion
Bioorganic Chemistry
Calcifediol
Catalysis
Chemistry
Chemistry and Materials Science
Cytochrome
Cytochrome P-450
Cytochrome P450
Gel electrophoresis
High performance liquid chromatography
Histology
Hydroxylase
Hydroxylation
Liquid chromatography
Metabolism
Morphology
NADP
Optimization
Organic Chemistry
Proteins
Sodium lauryl sulfate
Spectral analysis
Substrates
Vitamin D
Vitamin D3
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Summary: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.
ISSN:1572-3887
1573-4943
1875-8355
DOI:10.1007/s10930-018-9764-z