Data from: Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ~6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

酶的混杂性是生物催化剂快速趋异进化的必要前提。源自考斯图地芽孢杆菌（Geobacillus kaustophilus）HTA426的磷酸三酯酶样内酯酶（PLL）GkaP，兼具主要的内酯酶活性与混杂的磷酸三酯酶活性。为阐明其催化与进化机制，我们通过饱和诱变及X射线晶体学分析，对活性位点内的一处“热点”区域展开研究。结果发现，活性位点的99号位点参与底物识别。其中一株突变体Y99L对磷酸三酯酶底物乙基对氧磷的反应活性（kcat/Km）较野生型提升11倍，但对内酯酶底物δ-癸内酯的活性则下降15倍，最终实现了157倍的底物特异性反转。对Y99L的结构分析显示，该突变使相邻的7号环向外位移约6.6埃，这可能提升了活性位点的柔性，并促进有机磷酸酯底物的结合与/或催化。本研究为PLL家族提供了一个范例：仅需极少的突变即可实现从混杂性到专一性的进化路径，该类突变可改变活性位点环的构象调控，进而影响底物结合与酶活的能力。