Comparison of the stability of CYP105A1 and its variants engineered for production of active forms of vitamin D

Abstract

CYP105A1 from Streptomyces griseolus converts vitamin D3 to its biologically active form, 1α,25-dihydroxy D3. R73A/R84A mutation enhanced the 1α- and 25-hydroxylation activity for D3, while M239A mutation generated the 1α-hydroxylation activity for vitamin D2. In this study, the stability of six CYP105A1 enzymes, including five variants (R73A/R84A, M239A, R73A/R84A/M239A ( = TriA), TriA/E90A, and TriA/E90D), were examined. Circular dichroism analysis revealed that M239A markedly reduces the enzyme stability. Protein fluorescence analysis disclosed that these mutations, especially M239A, induce large changes in the local conformation around Trp residues. Strong stabilizing effect of glycerol was observed. Non-denaturing PAGE analysis showed that CYP105A1 enzymes are prone to self-association. Fluorescence analysis using a hydrophobic probe 8-anilino-1-naphthalenesulfonic acid suggested that M239A mutation enhances self-association and that E90A and E90D mutations, in cooperation with M239A, accelerate self-association with little effect on the stability.

Study on the Optimal Phospholipid Addition Levels for Button-Sized Juvenile Chinese Mitten Crab (Eriocheir sinensis): Survival, Growth, Physiological Parameters, and Neverland Expression

“…Nvd is verified to exert regulating effects by attaching to cholesterol 7-desaturase that dehydrogenates cholesterol to form 7-dehydrogenated cholesterol (the provitamin D), which was then catalyzed by cytochrome P450 (CYP) 105A1, and cytochrome P450 (CYP)2R1 to form vitamin D [29][30][31][32]. Vitamin D can promote the absorption of calcium and phosphorus of crustaceans and benefits their molting and growth [33][34][35].…”

“…Thus, it is believed that this gene plays a role in the transformation of cholesterol to 7-dehydrocholesterol [25][26][27][28]. Nvd is verified to exert regulating effects by attaching to cholesterol 7-desaturase that dehydrogenates cholesterol to form 7-dehydrogenated cholesterol (the provitamin D), which is successively catalyzed by cytochrome P450 (CYP) 105A1, and cytochrome P450 (CYP) 2R1 [29][30][31][32] to form 1α, 25-dihydroxy vitamin D 3 commonly known as vitamin D. Vitamin D can promote the absorption of calcium and phosphorus of crustaceans and benefits their molting and growth [33][34][35]. However, it remains unclear about the effects of PL addition on the expression of Nvd in E. sinensis.…”

Sarcopenia in liver cirrhosis: perspectives from epigenetics and microbiota

“…Some bacteria express enzymes involved in hydroxylation of steroids and are therefore able to process and activate vitamin D ( 74 ). Bacteria CYP105A1 from Streptomycesgriseolus converts vitamin D3 into 1,25(OH)2D3 in two independent reactions, suggesting that bacterial function is equivalent to that of vitamin D metabolizing enzymes ( 75 ). Therefore, rational regulation of the gut microbiota may promote vitamin D metabolism, increase VDR expression ( 76 ), and resist the effects of VDR gene hypermethylation.…”