Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Imizu, Toyama, Japan.
Biosci Biotechnol Biochem. 2022 Mar 21;86(4):444-454. doi: 10.1093/bbb/zbac019.
CYP105A1 from Streptomyces griseolus converts vitamin D3 to its biologically active form, 1α,25-dihydroxy vitamin D3. R73A/R84A mutation enhanced the 1α- and 25-hydroxylation activity for vitamin D3, while M239A mutation generated the 1α-hydroxylation activity for vitamin D2. In this study, the stability of six CYP105A1 enzymes, including 5 variants (R73A/R84A, M239A, R73A/R84A/M239A (=TriA), TriA/E90A, and TriA/E90D), was 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. Nondenaturing 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.
灰色链霉菌 CYP105A1 将维生素 D3 转化为具有生物活性的 1α,25-二羟基维生素 D3。R73A/R84A 突变增强了维生素 D3 的 1α-和 25-羟化活性，而 M239A 突变产生了维生素 D2 的 1α-羟化活性。在这项研究中，检测了包括 5 种突变体（R73A/R84A、M239A、R73A/R84A/M239A（=TriA）、TriA/E90A 和 TriA/E90D）在内的 6 种 CYP105A1 酶的稳定性。圆二色性分析表明，M239A 显著降低了酶的稳定性。蛋白质荧光分析揭示了这些突变，尤其是 M239A，诱导色氨酸残基周围局部构象发生了很大变化。甘油具有很强的稳定作用。非变性 PAGE 分析表明，CYP105A1 酶易于自身缔合。使用疏水性探针 8-苯胺基-1-萘磺酸的荧光分析表明，M239A 突变增强了自身缔合，而 E90A 和 E90D 突变与 M239A 合作，在几乎不影响稳定性的情况下加速自身缔合。
