Benfield Aaron P, Goodey Nina M, Phillips Lauren T, Martin Stephen F
Department of Chemistry and Biochemistry, The Institute of Cellular and Molecular Biology, The University of Texas, 1 University Station - A5300, Austin, TX 78712, USA.
Arch Biochem Biophys. 2007 Apr 1;460(1):41-7. doi: 10.1016/j.abb.2007.01.023. Epub 2007 Feb 12.
The phosphatidylcholine preferring phospholipase C from Bacillus cereus (PC-PLC(Bc)) catalyzes the hydrolysis of phospholipids in the following order of preference: phosphatidylcholine (PC)>phosphatidylethanolamine (PE)>phosphatidylserine (PS). In previous work, mutagenic, kinetic, and crystallographic experiments suggested that varying the amino acids at the 4th, 56th, and 66th positions had a significant influence upon the substrate specificity profile of PC-PLC(Bc). Here, we report the crystal structures of the native form of several PC-PLC(Bc) variants that exhibited altered substrate specificities for PC, PE, and PS at maximum resolutions of 1.90-2.05 Angstrom. Comparing the structures of these variants to the structure of the wild-type enzyme reveals only minor differences with respect to the number and location of active site water molecules and the side chain conformations of residues at the 4th and 56th positions. These results suggest that subtle changes in steric and electronic properties in the substrate binding site of PC-PLC(Bc) are responsible for the significant changes in substrate selectivity.
蜡样芽孢杆菌的磷脂酰胆碱偏好性磷脂酶C（PC-PLC(Bc)）催化磷脂水解的偏好顺序如下：磷脂酰胆碱（PC）>磷脂酰乙醇胺（PE）>磷脂酰丝氨酸（PS）。在之前的工作中，诱变、动力学和晶体学实验表明，改变第4、56和66位的氨基酸对PC-PLC(Bc)的底物特异性谱有显著影响。在此，我们报道了几种PC-PLC(Bc)变体天然形式的晶体结构，这些变体对PC、PE和PS表现出改变的底物特异性，最高分辨率为1.90 - 2.05埃。将这些变体的结构与野生型酶的结构进行比较，发现在活性位点水分子的数量和位置以及第4和56位残基的侧链构象方面只有微小差异。这些结果表明，PC-PLC(Bc)底物结合位点的空间和电子性质的细微变化是底物选择性显著变化的原因。
