School of Environmental & Biological Engineering, Nanjing University of Science and Technology, Xiaolingwei 200, Nanjing, China, 210094.
Int J Biol Sci. 2019 Jan 1;15(2):341-350. doi: 10.7150/ijbs.28904. eCollection 2019.
UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid by NAD-dependent two-fold oxidation. Despite extensive investigation into the catalytic mechanism of UGDH, the previously proposed mechanisms regarding the first-step oxidation are somewhat controversial and inconsistent with some biochemical evidence, which instead supports a mechanism involving an NAD-dependent bimolecular nucleophilic substitution (S2) reaction. To verify this speculation, the essential Cys residue of UGDH (UGDH) was changed to an Ala residue, and the resulting Cys260Ala mutant and UGDH were then co-expressed via a single-crossover homologous recombination method. Contrary to the previously proposed mechanisms, which predict the formation of the capsular polysaccharide hyaluronan, the resulting strain instead produced an amide derivative of hyaluronan, as validated via proteinase K digestion, ninhydrin reaction, FT-IR and NMR. This result is compatible with the NAD-dependent S2 mechanism.
UDP-葡萄糖脱氢酶（UGDH）通过 NAD 依赖性的两倍氧化作用催化 UDP-葡萄糖向 UDP-葡萄糖醛酸的转化。尽管对 UGDH 的催化机制进行了广泛的研究，但先前提出的关于第一步氧化的机制有些争议，并且与一些生化证据不一致，这些证据反而支持涉及 NAD 依赖性双分子亲核取代（S2）反应的机制。为了验证这一推测，将 UGDH（UGDH）的必需半胱氨酸残基突变为丙氨酸残基，然后通过单交叉同源重组方法共表达所得的 Cys260Ala 突变体和 UGDH。与先前提出的机制相反，该机制预测了荚膜多糖透明质酸的形成，而所得菌株反而产生了透明质酸的酰胺衍生物，这通过蛋白酶 K 消化、茚三酮反应、FT-IR 和 NMR 得到了验证。这一结果与 NAD 依赖性 S2 机制兼容。
