The Mycobacterium DprE2 is a NADH-dependent enzyme and converts the decaprenylphosphoryl-beta-D-ribose (DPX) to decaprenylphosphoryl-β-D-arabinofuranose (DPA). The FAD-containing oxidoreductase MtbDprE1 and NADH-dependent reductase MtbDprE2 enzymes catalyses together the epimerization reaction, which coverts DPR to DPA. Here, MtbDprE2 enzyme was purified and structurally characterized using circular dichroism, molecular modelling and dynamics simulation techniques. The MtbDprE2 was purified, which eluted as oligomer from size exclusion column. The circular dichroism analysis yielded ~ 47.6% alpha-helix, ~ 19.8% beta-sheet and ~ 32.6% random coil structures in MtbDprE2 enzyme and showed highly thermostability. The molecular modelling of MtbDprE2 and its complex with NADH showed that it contains two domains (i) the large domain consists of central twisted seven beta-sheets decorated by eight alpha-helices and (ii) a small domain contains two short alpha-helices connect by loop. Overall, the MtbDprE2 adopts a typical short-chain dehydrogenase rossmann fold and NADH binds to Asp69, Ser147, Tyr160, Lys164 of catalytic triad and Gly16, Ser19, Glu20, Ile21 of Gly-rich motif of MtbDprE2. 1 ns dynamics simulation was performed on apo and NADH bound MtbDprE2, which indicated the small conformational change in ligand binding site, which resulted more closed pocket than open pocket observed in apo enzyme. Small conformational changes were observed in active site residues and orientation between large and small domains of MtbDprE2 upon NADH binding. Current knowledge of MtbDprE2 structure and its NADH binding mechanism will contribute significantly in development of specific inhibitors against M. tuberculosis.

中文翻译：

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结核分枝杆菌癸二烯基磷酸基-β-D-核糖差向异构酶-2酶参与细胞壁生物发生的结构研究

分枝杆菌DprE2是NADH依赖性酶，可将癸烯基磷酰基-β-D-核糖（DPX）转化为癸烯基磷酰基-β-D-阿拉伯呋喃糖（DPA）。含FAD的氧化还原酶MtbDprE1和NADH依赖性还原酶MtbDprE2酶共同催化差向异构反应，使DPR转化为DPA。在这里，MtbDprE2酶经过纯化，并使用圆二色性，分子建模和动力学模拟技术对其结构进行了表征。纯化MtbDprE2，将其作为寡聚物从尺寸排阻柱洗脱。圆二色性分析在MtbDprE2酶中产生了约47.6％的α-螺旋，约19.8％的β-折叠和约32.6％的无规卷曲结构，并显示出高度的热稳定性。MtbDprE2及其与NADH的复合物的分子模型研究表明，它包含两个域（i）大域由中央扭曲的七个由八个α-螺旋修饰的β-折叠组成，（ii）一个小域包含两个短α-螺旋连接通过循环。总体而言，MtbDprE2采用典型的短链脱氢酶罗斯曼折叠，NADH结合催化​​三联体的Asp69，Ser147，Tyr160，Lys164和MtbDprE2富含Gly基序的Gly16，Ser19，Glu20，Ile21。对载脂蛋白和NADH结合的MtbDprE2进行了1 ns的动力学模拟，这表明配体结合位点的构象变化很小，导致在载脂蛋白酶中观察到的封闭口袋比开放口袋更多。观察到NADH结合后，MtbDprE2的活性位点残基和大，小结构域之间的取向发生了小的构象变化。