State Key Laboratory of Food Nutrition and Safety, Tianjin, People's Republic of China.
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin, People's Republic of China.
Microb Cell Fact. 2018 Sep 10;17(1):141. doi: 10.1186/s12934-018-0981-0.
Biosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor.
Recombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (k/K) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively).
The successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity.
与化学合成相比，甾体药物的生物合成在制药生产中具有很大的优势，因为该过程涉及在环境温度和大气压下进行高效的酶催化。节杆菌（Arthrobacter simplex）中的 3-酮甾体-∆-脱氢酶（KsdD3）以 FAD 为辅因子催化甾体底物的 1,2-去饱和作用。
重组 KsdD3 表现出有机溶剂耐受性。位于底物结合腔中的 W117、F296、W299 等残基被预测与底物形成疏水相互作用。对 KsdD3 进行基于结构的定点饱和突变，得到了对各种甾体底物具有更高催化活性的突变体。与野生型酶相比，W299A 显示出最高的催化效率（k/K）增加。同源建模表明，突变体扩大了活性位点腔，减轻了空间位阻干扰，有利于识别 C17 羟基/羰基甾体底物。定向分子动力学模拟表明，W299A/G 降低了底物结合和相应产物解离的势能垒。用酶催化和携带 KsdD3 WT/突变体的静止细胞进行 AD 的生物转化表明，与野生型（分别为 38%和 75%）相比，W299A 分别通过酶催化和静止细胞转化催化 ADD 产率最高，分别为 71%和 95%。
成功的功能 KsdD3 合理设计极大地提高了我们对 KsdD 家族酶的理解。基于结构的定点饱和突变和生化数据用于设计具有更高催化活性和更广泛选择性的 KsdD3 突变体。
