Characterization and crystal structure of Escherichia coli KDPGal aldolase.
Characterization and crystal structure of Escherichia coli KDPGal aldolase.
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大肠杆菌 KDPGal 醛缩酶的表征和晶体结构。
DOI：
10.1016/j.bmc.2007.10.043
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发表时间：
2008
影响因子：
3.5
通讯作者：
Toone,EricJ
中科院分区：
文献类型：
--
作者：
Walters,MatthewJ;Srikannathasan,Velupillai;McEwan,AndrewR;Naismith,JamesH;Fierke,CarolA;Toone,EricJ
2-酮-3-脱氧-6-磷酸葡萄糖酸（KDPG）和2-酮-3-脱氧-6-磷酸半乳糖酸（KDPGal）醛缩酶催化相同的反应，不同之处仅在于单个立体中心的构型的底物特异性。然而，蛋白质在氨基酸水平上显示出很少的序列同源性。在这里，我们调查这些酶的底物选择性的决定因素。将大肠杆菌KDPGal醛缩酶基因克隆到T7表达载体中，并在E.大肠杆菌中，催化天然底物KDPGal的逆醛醇裂解，其kcat/Km和kcat值分别为1.9× 104 M − 1 s − 1和4s−1。在合成方向上，KDPGal醛缩酶使用有限数量的醛底物有效地催化羟醛加成，所述醛底物包括d-甘油醛-3-磷酸（天然底物）、d-甘油醛、乙醇醛和2-吡啶甲醛。由KDPGal醛缩酶催化的2-吡啶甲醛和丙酮酸之间的制备规模反应以> 99.7%ee产生R立体化学的羟醛加合物，这一结果与使用相关KDPG醛缩酶观察到的结果互补。天然晶体结构的解析度为2.4 μ m，显示出与KDPG醛缩酶相同的（α/β）8拓扑结构。我们还确定了一个2.1 π结构的Schiff碱复合物之间的酶和它的底物。该模型预测，一个单一的氨基酸的变化，T161在KDPGal醛缩酶的KDPGal醛缩酶的V154，起着重要的作用，在确定酶催化的立体化学过程中，这一预测证实了定点诱变研究。然而，需要对酶序列进行额外的改变才能制备具有高催化效率和改变的立体化学的酶。
2-Keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases catalyze an identical reaction differing in substrate specificity in only the configuration of a single stereocenter. However, the proteins show little sequence homology at the amino acid level. Here we investigate the determinants of substrate selectivity of these enzymes. The Escherichia coli KDPGal aldolase gene, cloned into a T7 expression vector and overexpressed in E. coli, catalyzes retro-aldol cleavage of the natural substrate, KDPGal, with values of kcat/KMand kcatof 1.9×104M−1s−1and 4s−1, respectively. In the synthetic direction, KDPGal aldolase efficiently catalyzes an aldol addition using a limited number of aldehyde substrates, including d-glyceraldehyde-3-phosphate (natural substrate), d-glyceraldehyde, glycolaldehyde, and 2-pyridinecarboxaldehyde. A preparative scale reaction between 2-pyridinecarboxaldehyde and pyruvate catalyzed by KDPGal aldolase produced the aldol adduct of the R stereochemistry in >99.7% ee, a result complementary to that observed using the related KDPG aldolase. The native crystal structure has been solved to a resolution of 2.4Å and displays the same (α/β)8topology, as KDPG aldolase. We have also determined a 2.1Å structure of a Schiff base complex between the enzyme and its substrate. This model predicts that a single amino acid change, T161 in KDPG aldolase to V154 in KDPGal aldolase, plays an important role in determining the stereochemical course of enzyme catalysis and this prediction was borne out by site-directed mutagenesis studies. However, additional changes in the enzyme sequence are required to prepare an enzyme with both high catalytic efficiency and altered stereochemistry.