通过结构引导工程改造荧光假单胞菌甘露醇2-脱氢酶的辅酶特异性，以实现对NAD(H)和NADP(H)的高效利用。

Structure-guided engineering of the coenzyme specificity of Pseudomonas fluorescens mannitol 2-dehydrogenase to enable efficient utilization of NAD(H) and NADP(H).

作者信息

Bubner Patricia, Klimacek Mario, Nidetzky Bernd

机构信息

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, Graz, Austria.

出版信息

FEBS Lett. 2008 Jan 23;582(2):233-7. doi: 10.1016/j.febslet.2007.12.008. Epub 2007 Dec 17.

DOI:10.1016/j.febslet.2007.12.008

PMID:18082142

Abstract

The structure of Pseudomonas fluorescens mannitol 2-dehydrogenase with bound NAD+ leads to the suggestion that the carboxylate group of Asp(69) forms a bifurcated hydrogen bond with the 2' and 3' hydroxyl groups of the adenosine of NAD+ and contributes to the 400-fold preference of the enzyme for NAD+ as compared to NADP+. Accordingly, the enzyme with the Asp(69)-->Ala substitution was found to use NADP(H) almost as well as wild-type enzyme uses NAD(H). The Glu(68)-->Lys substitution was expected to enhance the electrostatic interaction of the enzyme with the 2'-phosphate of NADP+. The Glu(68)-->Lys:Asp(69)-->Ala doubly mutated enzyme showed about a 10-fold preference for NADP(H) over NAD(H), accompanied by a small decrease in catalytic efficiency for NAD(H)-dependent reactions as compared to wild-type enzyme.

摘要

结合NAD⁺的荧光假单胞菌甘露醇2-脱氢酶的结构表明，Asp(69)的羧基与NAD⁺腺苷的2'和3'羟基形成分叉氢键，并且与NADP⁺相比，该酶对NAD⁺的偏好性高400倍。因此，发现Asp(69)被替换为Ala的酶使用NADP(H)的能力几乎与野生型酶使用NAD(H)的能力相同。预期Glu(68)被替换为Lys会增强酶与NADP⁺的2'-磷酸的静电相互作用。Glu(68)被替换为Lys、Asp(69)被替换为Ala的双突变酶对NADP(H)的偏好性比对NAD(H)高约10倍，与野生型酶相比，其NAD(H)依赖性反应的催化效率略有降低。