Biochemical Characterization of the Cytochrome P450 CYP107CB2 from Bacillus lehensis G1

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.

Expression and Characterization of a New Self‐Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans

“…P450DA from Deinococcus apachensis showed maximum activity at 20 °C and pH 7.5 phosphate buffer [42] . Similarly, CYP107CB2 from Bacillus lehensis G1 showed the highest activity towards vitamin D3 at 25 °C and pH 8.0 in phosphate buffer [43] . Hal1 from Halomonas sp.…”

“…[42] Similarly, CYP107CB2 from Bacillus lehensis G1 showed the highest activity towards vitamin D3 at 25 °C and pH 8.0 in phosphate buffer. [43] Hal1 from Halomonas sp. NCIMB 172 displayed a halflife of 1 h when incubated at 30 °C in tricine buffer at pH 8.5.…”

Vitamins as regulators of calcium-containing kidney stones — new perspectives on the role of the gut microbiome

“…Specifically, multiple environmental bacteria in the Actinomycetia class possess a cytochrome P-450 (CYP) enzyme capable of converting vitamin D into 25(OH)D (storage form) or 1,25(OH) 2 D (active form) 204 – 206 . The soil isolate Bacillus lehensis G1 was also shown to possess CYP enzymes that can convert vitamin D 3 into 25(OH)D 3 , suggesting that this function is present in multiple lineages of bacteria 207 , 208 . The genes encoding these unique CYP enzymes have not been found in the genomic pool of human intestinal bacteria; however, a protein having partial homology with human CYP27A1 was identified in Ruminococcus torques , which is part of the human gut microbiota 209 .…”

Bioconversion of vitamin D3 to bioactive calcifediol and calcitriol as high-value compounds

“…In addition, CYP105A1 can also catalyze the hydroxylation of VD 2 with a relatively high V max /K m value (0.142 L −1 •min −1 /mol P450, compared to 30 L −1 •min −1 /mol P450 for VD 3 ). The CYP107CB2 from Bacillus lehensis G1 was classified into the CYP107 subfamily and showed 25-hydroxylation activity of both VD 3 and 1α(OH)VD 3 [42].…”