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首页> 外文期刊>Biochemistry >Optimizing an Artificial Metabolic Pathway: Engineering the Cofactor Specificity of Corynebacterium 2,5-Diketo-D-gluconic Acid Reductase for Use in Vitamin C Biosynthesis
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Optimizing an Artificial Metabolic Pathway: Engineering the Cofactor Specificity of Corynebacterium 2,5-Diketo-D-gluconic Acid Reductase for Use in Vitamin C Biosynthesis

机译：优化人工代谢途径：设计用于维生素C生物合成的棒杆菌2，5-二酮-D-葡萄糖酸还原酶的辅因子特异性

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摘要

The strict cofactor specificity of many enzymes can potentially become a liability when these enzymes are to be employed in an artificial metabolic pathway. The preference for NADPH over NADH exhibited by the Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase may not be ideal for use in industrial scale vitamin C biosynthesis. We have previously reported making a number of sitedirected mutations at five sites located in the cofactor-binding pocket that interact with the 2'-phosphate group of NADPH. These mmutations conferred greater activity with NADH upon the Corynebacterium 2,5-DKG reductase [Banta, S., Swanson, B. A., Wu, S., Jarnagin, A., and Anderson, S. (2002) Protein Eng. 15, 131-140; (1)]. The best of these mutations have now vbeen combined to see if further improvements can be obtained. In addition, several chimeric mutants have been produced that contain the same residues as are found in other members of the aldo-keto reductase superfamily that are naturally able to use NADH as a cofactor. The most active mutants obtained in this work were also combined with a previously reported substrate-binding pocket double mutant, F22Y/A272G. Mutant activity was assayed using activity-stained native polyacrylamide gels. Superior mutants were purified and subjected to a simplified kinetic analysis. The simplified kinetic analysis was extended for the most active mutants in order to obtain the kinetic parameters in the full-ordered bi bi rate equation in the absence of products, with both NADH and NADPH as cofactors. The best mutant 2,5-DKG reductase produced in this work was the F22Y/K232G/R238H/A272G quadruple mutant, which exhibits activity with NADH that is more than 2 orders of magnitude higher than that of the wild-type enayme, and it retains a high level of activity with NADPH. This new 2,5-DKG reductase may be a valuable new catalyst for use in vitamin C biosynthesis.
机译：当将这些酶用于人工代谢途径中时，许多酶的严格的辅因子特异性可能成为责任。 NADPH优于棒杆菌2,5-二酮-D-葡萄糖酸（2,5-DKG）还原酶表现出的NADH可能不适用于工业规模的维生素C生物合成。我们以前曾报道过在与NADPH的2'-磷酸基团相互作用的辅因子结合袋中的五个位点发生了许多定点突变。这些突变赋予NADH对棒杆菌2，5-DKG还原酶更大的活性[Banta，S.，Swanson，B.A.，Wu，S.，Jarnagin，A。，和Anderson，S。（2002）Protein Eng.Chem.Soc.Sci。，Vol.5，pp.5-8。 15、131-140; （1）]。现在已经结合了这些突变中最好的一种，以查看是否可以获得进一步的改进。另外，已经产生了一些嵌合突变体，其含有与在醛糖-酮还原酶超家族的其他成员中发现的相同的残基，这些残基自然能够使用NADH作为辅因子。在这项工作中获得的最活跃的突变体还与先前报道的底物结合口袋双突变体F22Y / A272G结合。使用活性染色的天然聚丙烯酰胺凝胶测定突变活性。纯化高级突变体并进行简化的动力学分析。简化了动力学分析，对活性最高的突变体进行了扩展，以便在不存在产物的情况下，以NADH和NADPH为辅因子，在全序bibi速率方程中获得动力学参数。这项工作中产生的最佳突变体2,5-DKG还原酶是F22Y / K232G / R238H / A272G四重突变体，它对NADH的活性比野生型酶高2个数量级，保持NADPH的高水平活性。这种新的2,5-DKG还原酶可能是用于维生素C生物合成的有价值的新催化剂。

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