2-keto-3-L-arabinonate dehydratase (L-KdpD) and 2-keto-3-D-xylonate dehydratase (D-KdpD) are the third enzymes in the Weimberg pathway catalyzing the dehydration of respective 2-keto-3-deoxy sugar acids (KDP) to α-ketoglutaric semialdehyde (KGSA). The Weimberg pathway has been explored recently with respect to the synthesis of chemicals from L-arabinose and D-xylose. However, only limited work has been done toward characterizing these two enzymes. In this work, several new L-KdpDs and D-KdpDs were cloned and heterologously expressed in Escherichia coli. Following kinetic characterizations and kinetic stability studies, the L-KdpD from Cupriavidus necator (CnL-KdpD) and D-KdpD from Pseudomonas putida (PpD-KdpD) appeared to be the most promising variants from each enzyme class. Magnesium had no effect on CnL-KdpD, whereas increased activity and stability were observed for PpD-KdpD in the presence of Mg2+. Furthermore, CnL-KdpD was not inhibited in the presence of L-arabinose and L-arabinonate, whereas PpD-KdpD was inhibited with D-xylonate (I50 of 75 mM), but not with D-xylose. Both enzymes were shown to be highly active in the one-step conversions of L-KDP and D-KDP. CnL-KdpD converted > 95% of 500 mM L-KDP to KGSA in the first 2 h while PpD-KdpD converted > 90% of 500 mM D-KDP after 4 h. Both enzymes in combination were able to convert 83% of a racemic mixture of D,L-KDP (500 mM) after 4 h, with both enzymes being specific toward the respective stereoisomer. Key points • L-KdpDs and D-KdpDs are specific toward L- and D-KDP, respectively. • Mg2+affected activity and stabilities of D-KdpDs, but not of L-KdpDs. • CnL-KdpD and PpD-KdpD converted 0.5 M of each KDP isomer reaching 95 and 90% yield. • Both enzymes in combination converted 0.5 M racemic D,L-KDP reaching 83% yield.

中文翻译：

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高活性 2-酮-3-脱氧-L-阿拉伯酸和 2-酮-3-脱氧-D-木糖酸脱水酶在半纤维素糖生物转化为化学品方面的表征。


2-酮-3-L-阿拉伯酸脱水酶 (L-KdpD) 和 2-酮-3-D-木糖酸脱水酶 (D-KdpD) 是 Weimberg 途径中的第三种酶，催化各自 2-酮-3- 脱水。脱氧糖酸（KDP）转化为α-酮戊二酸半醛（KGSA）。最近，人们对 Weimberg 途径进行了关于从 L-阿拉伯糖和 D-木糖合成化学物质的探索。然而，在表征这两种酶方面只做了有限的工作。在这项工作中，几个新的L-KdpD和D-KdpD被克隆并在大肠杆菌中异源表达。经过动力学表征和动力学稳定性研究，来自 Cupriavidus necator 的 L-KdpD (CnL-KdpD) 和来自恶臭假单胞菌的 D-KdpD (PpD-KdpD) 似乎是每个酶类别中最有前途的变体。镁对 CnL-KdpD 没有影响，而在 Mg2+ 存在下观察到 PpD-KdpD 的活性和稳定性增加。此外，CnL-KdpD 在 L-阿拉伯糖和 L-阿拉伯糖酸盐存在下不受抑制，而 PpD-KdpD 在 D-木糖酸盐存在下受到抑制（I50 为 75 mM），但在 D-木糖存在下则不受抑制。两种酶在 L-KDP 和 D-KDP 的一步转化中均表现出高度活性。 CnL-KdpD 在前 2 小时内将 > 95% 的 500 mM L-KDP 转化为 KGSA，而 PpD-KdpD 在 4 小时后将 > 90% 的 500 mM D-KDP 转化为 KGSA。 4 小时后，两种酶组合能够转化 83% 的 D,L-KDP (500 mM) 外消旋混合物，并且两种酶对各自的立体异构体具有特异性。要点 • L-KdpD 和D-KdpD 分别针对L-和D-KDP。 • Mg2+ 影响D-KdpDs 的活性和稳定性，但不影响L-KdpDs。 • CnL-KdpD 和PpD-KdpD 转化了每种KDP 异构体0.5 M，产率达到95% 和90%。 • 两种酶组合后转化为 0。5M外消旋D,L-KDP收率达到83%。