School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.
PLoS Comput Biol. 2012;8(7):e1002612. doi: 10.1371/journal.pcbi.1002612. Epub 2012 Jul 12.
Control of enzyme allosteric regulation is required to drive metabolic flux toward desired levels. Although the three-dimensional (3D) structures of many enzyme-ligand complexes are available, it is still difficult to rationally engineer an allosterically regulatable enzyme without decreasing its catalytic activity. Here, we describe an effective strategy to deregulate the allosteric inhibition of enzymes based on the molecular evolution and physicochemical characteristics of allosteric ligand-binding sites. We found that allosteric sites are evolutionarily variable and comprised of more hydrophobic residues than catalytic sites. We applied our findings to design mutations in selected target residues that deregulate the allosteric activity of fructose-1,6-bisphosphatase (FBPase). Specifically, charged amino acids at less conserved positions were substituted with hydrophobic or neutral amino acids with similar sizes. The engineered proteins successfully diminished the allosteric inhibition of E. coli FBPase without affecting its catalytic efficiency. We expect that our method will aid the rational design of enzyme allosteric regulation strategies and facilitate the control of metabolic flux.
为了将代谢通量推向所需水平，需要控制酶变构调节。尽管许多酶-配体复合物的三维（3D）结构已经可用，但如果不降低其催化活性，仍然很难合理设计可变构调节的酶。在这里，我们描述了一种基于变构配体结合位点的分子进化和物理化学特性来解除酶变构抑制的有效策略。我们发现变构位点在进化上是可变的，并且包含比催化位点更多的疏水性残基。我们将这些发现应用于设计选定靶标残基中的突变，以解除果糖-1,6-二磷酸酶（FBPase）的变构活性。具体来说，在不太保守的位置的带电荷氨基酸被疏水性或中性氨基酸取代，大小相似。工程蛋白成功地减轻了大肠杆菌 FBPase 的变构抑制，而不影响其催化效率。我们期望我们的方法将有助于酶变构调节策略的合理设计，并促进代谢通量的控制。
