Comparison of the stability of CYP105A1 and its variants engineered for production of active forms of vitamin D
Comparison of the stability of CYP105A1 and its variants engineered for production of active forms of vitamin D
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CYP105A1 及其设计用于生产活性维生素 D 形式的变体的稳定性比较
DOI：
10.1093/bbb/zbac019
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Yasukawa Kiyoshi
中科院分区：
文献类型：
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作者：
Takita Teisuke;Sakuma Hiro;Ohashi Ren;Nilouyal Somaye;Nemoto Sho;Wada Moeka;Yogo Yuya;Yasuda Kaori;Ikushiro Shinichi;Sakaki Toshiyuki;Yasukawa Kiyoshi
来自灰色链霉菌的CYP 105 A1将维生素D3转化为其生物活性形式1α，25-二羟基维生素D3。R73 A/R84 A突变增强维生素D3的1α-和25-羟基化活性，而M239 A突变产生维生素D2的1α-羟基化活性。在本研究中，检查了6种CYP 105 A1酶的稳定性，包括5种变体（R73 A/R84 A、M239 A、R73 A/R84 A/M239 A（=TriA）、TriA/E90 A和TriA/E90 D）。圆二色谱分析表明，M239 A显着降低酶的稳定性。蛋白质荧光分析显示，这些突变，特别是M239 A，诱导周围的色氨酸残基的局部构象的大的变化。观察到甘油的强稳定作用。非变性PAGE分析表明，CYP 105 A1酶易于自缔合。使用疏水探针8-anilino-1-naphthalenesulfonic acid的荧光分析表明，M239 A突变增强了自缔合，并且E90 A和E90 D突变与M239 A合作，加速了自缔合，对稳定性影响不大。
CYP105A1 fromStreptomyces griseolusconverts 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.