Institute of Microbiology, Universität Stuttgart, Allmandring 31, Germany.
Chembiochem. 2010 Mar 22;11(5):681-90. doi: 10.1002/cbic.200900720.
Recently, we reported on a transaldolase B variant (TalB F178Y) that is able to use dihydroxyacetone (DHA) as donor in aldol reactions. In a second round of protein engineering, we aimed at improving the affinity of this variant towards nonphosphorylated acceptor aldehydes, that is, glyceraldehyde (GA). The anion binding site was identified in the X-ray structure of TalB F178Y where a sulfate ion from the buffer was bound in the active site. Therefore, we performed site-directed saturation mutagenesis at three residues forming the putative phosphate binding site, Arg181, Ser226 and Arg228. The focused libraries were screened for the formation of D-fructose from DHA and d,l-GA by using an adjusted colour assay. The best results with respect to the synthesis of D-fructose were achieved with the TalB F178Y/R181E variant, which exhibited an at least fivefold increase in affinity towards d,l-GA (K(M)=24 mM). We demonstrated that this double mutant can use D-GA, glycolaldehyde and the L-isomer, L-GA, as acceptor substrates. This resulted in preparative synthesis of D-fructose, D-xylulose and L-sorbose when DHA was used as donor. Hence, we engineered a DHA-dependent aldolase that can synthesise the formation of polyhydroxylated compounds from simple and cheap substrates at preparative scale.
最近，我们报道了一种转醛醇酶 B 变体（TalB F178Y），它能够将二羟丙酮（DHA）用作醛醇反应的供体。在第二轮蛋白质工程中，我们旨在提高该变体对非磷酸化受体醛的亲和力，即甘油醛（GA）。阴离子结合位点在 TalB F178Y 的 X 射线结构中被鉴定出来，其中缓冲液中的硫酸根离子结合在活性位点中。因此，我们在可能的磷酸结合位点的三个残基处进行了定点饱和突变，Arg181、Ser226 和 Arg228。通过使用调整后的显色测定法，从 DHA 和 d,l-GA 合成 D-果糖来筛选聚焦文库。在 TalB F178Y/R181E 变体中，与 d,l-GA 的亲和力至少提高了五倍（K(M)=24 mM），在合成 D-果糖方面取得了最佳结果。我们证明了这种双突变体可以将 D-GA、乙二醇醛和 L-异构体 L-GA 用作受体底物。当 DHA 用作供体时，这导致了 D-果糖、D-木酮糖和 L-山梨糖的制备性合成。因此，我们设计了一种依赖 DHA 的醛醇酶，它可以从简单廉价的底物在制备规模上合成多羟基化合物。
