铜绿假单胞菌中PA0847二鸟苷酸环化酶活性受非催化位点Y700调控

The non-catalytic site Y700 regulates diguanylate cyclase activity of PA0847 in Pseudomonas aeruginosa

【目的】鉴定铜绿假单胞菌(Pseudomonas aeruginosa)中PA0847胞内结构域及其突变体的二鸟苷酸环化酶活性,并初步探究其催化机制。【方法】利用刚果红平板染色法验证PA0847胞内域的二鸟苷酸环化酶活性;采用PCR技术构建PA0847 PAS-GGDEF结构域及其单点突变体,并经过表达纯化获得相应蛋白;经凝胶分子筛层析分析蛋白在溶液中的聚集状态;通过体外酶促反应鉴定蛋白的二鸟苷酸环化酶活性,基于噻唑橙荧光染色检测蛋白酶促反应后环鸟苷二磷酸(cyclic diguanylate monophosphate,c-di-GMP)的生成量,并筛选与二鸟苷酸环化酶活性密切相关的氨基酸残基;联用结构预测和分子对接获得PA0847 PAS-GGDEF二聚体以及结合三磷酸鸟苷(guanosine triphosphate,GTP)的复合物结构模型。【结果】PA0847 PAS-GGDEF主要以二聚体形式发挥催化活性,PAS结构域促进二聚体形成,有效提高二鸟苷酸环化酶活性;突变筛选发现,在低蛋白浓度(0.6 mg/m L)下,非催化位点突变体Y700A活性较野生型显著提高;凝胶分子筛层析表明,其高活性可能与Y700A突变促进GGDEF(Gly-Gly-Asp-Glu-Phe)结构域二聚化有关;结构模型分析显示,PA0847 PAS-GGDEF与GTP结合位点保守,其中K722的氨基侧链在结合GTP的磷酸基团中发挥着重要作用,而Y700芳香环侧链与K722所在的α螺旋有疏水互作,因此Y700A突变可能改变了K722所在螺旋的空间取向,进而促进K722与底物GTP的结合以及GGDEF二聚化。【结论】铜绿假单胞菌PA0847的非催化位点Y700可间接调控二鸟苷酸环化酶活性。

[Objective]To identify the diguanylate cyclase activity of the intracellular domain and its mutants of PA0847 from Pseudomonas aeruginosa and preliminarily probe into its catalytic mechanism.[Methods]Congo red plate staining was employed to verify the diguanylate cyclase activity of the intracellular domain of PA0847.PCR was employed to construct the PA0847 PAS-GGDEF domain and its single-point mutants,and the corresponding proteins were expressed and purified.Gel filtration chromatography was utilized to analyze the aggregation states of proteins in solution.The diguanylate cyclase activity of the proteins was identified by in-vitro enzymatic reactions.Based on thiazole orange fluorescence staining,the production of cyclic diguanylate monophosphate(c-di-GMP)was determined after the enzymatic reactions,and the amino acid residues closely related to the activity of diguanylate cyclase were screened.The structural models of PA0847 PAS-GGDEF and its complex with guanosine triphosphate(GTP)were obtained by structure prediction combined with molecular docking.[Results]PA0847 PAS-GGDEF primarily exerted its catalytic activity as a dimer,with the PAS domain facilitating the dimer formation and increasing the activity of the diguanylate cyclase.Mutant screening revealed a significant increase in the activity of the non-catalytic site mutant Y700A compared with the wild type at a low protein concentration(<0.6 mg/mL).Gel filtration chromatography indicated that the heightened activity may be attributed to the enhanced GGDEF(Gly-Gly-Asp-Glu-Phe)dimerization driven by Y700A.Structural modeling revealed that PA0847 PAS-GGDEF had a conserved GTP binding site,in which the amino side chain of K722 played an important role in binding to the phosphoryl group of GTP.The aromatic ring of Y700 engaged in a hydrophobic interaction with the alpha-helix containing K722.Therefore,the mutation Y700A may alter the spatial orientation of the K722-containing helix,which promoted the binding of K722 to the substrate GTP and the dimerization of GGDEF.[Conclusion]The non-catalytic site Y700 of PA0847 from Pseudomonas aeruginosa can indirectly regulate the diguanylate cyclase activity.