Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A
Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A
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DOI：
10.1093/protein/15.2.131
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发表时间：
2002-02-01
期刊：
影响因子：
--
通讯作者：
Anderson, S
中科院分区：
文献类型：
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作者：
Banta, S;Swanson, BA;Anderson, S
NADPH依赖性2，5-二酮-D-葡萄糖酸（2，5-DKG）还原酶是一些新型生物合成维生素C生产过程中的必需组分。这种酶催化2，5-DKG转化为2-酮基-L-古洛糖酸，后者是L-抗坏血酸的直接前体。在2，5-DKG还原酶A的辅因子结合口袋中的5个残基处进行了40个独特的定点突变，试图提高其使用NADH作为辅因子的能力。NADH比NADPH更稳定，更便宜，在细胞中更普遍。据我们所知，这是第一次集中尝试改变辅因子特异性的醛酮还原酶超家族的成员，通过工程改造的活性与NADH到酶。使用活性染色的天然聚丙烯酰胺凝胶测定具有NADH或NADPH的突变体的活性。在三个不同位点的突变体中有八个被鉴定为具有改进的NADH活性。这些突变体进行纯化，并进行动力学表征与NADH作为辅因子。获得的最佳突变体，R238 H，与野生型酶相比，产生了几乎7倍的改进与NADH的催化作用。令人惊讶的是，这种催化改善的大部分似乎是由于反应的表观k（cat）的改善，而不是酶对NADH的亲和力的大幅改善。
The NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme is a required component in some novel biosynthetic vitamin C production processes. This enzyme catalyzes the conversion of 2,5-DKG to 2-keto-L-gulonic acid, which is an immediate precursor to L-ascorbic acid. Forty unique site-directed mutations were made at five residues in the cofactor-binding pocket of 2,5-DKG reductase A in an attempt to improve its ability to use NADH as a cofactor. NADH is more stable, less expensive and more prevalent in the cell than is NADPH. To the best of our knowledge, this is the first focused attempt to alter the cofactor specificity of a member of the aldo-keto reductase superfamily by engineering improved activity with NADH into the enzyme. Activity of the mutants with NADH or NADPH was assayed using activity-stained native polyacrylamide gels. Eight of the mutants at three different sites were identified as having improved activity with NADH. These mutants were purified and subjected to a kinetic characterization with NADH as a cofactor. The best mutant obtained, R238H, produced an almost 7-fold improvement in catalysis with NADH compared with the wild-type enzyme. Surprisingly, most of this catalytic improvement appeared to be due to an improvement in the apparent k(cat) for the reaction rather than a large improvement in the affinity of the enzyme for NADH.