Bubner Patricia, Klimacek Mario, Nidetzky Bernd
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, Graz, Austria.
FEBS Lett. 2008 Jan 23;582(2):233-7. doi: 10.1016/j.febslet.2007.12.008. Epub 2007 Dec 17.
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.
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。
