新型YdjM超家族成员的钠/氢逆向转运蛋白功能鉴定

Functional Identification of Na^+/H^+ Antiporter in Novel YdjM Superfamily Members

在原核生物中,钠/氢逆向转运蛋白具有催化细胞内的Na^+、Li^+或K^+等碱基阳离子的排出,换取外部质子,以降低有毒碱性金属阳离子的细胞质浓度和维持细胞内pH稳态起到了至关重要的作用。为了进一步挖掘中度嗜盐菌HalobacillusY5中具有盐碱耐受性的钠/氢逆向转运蛋白基因并对其功能进行鉴定,我们首先提取该菌的基因组DNA,然后采用Sau3AI随机酶切及功能互补的方法获得了一个新型的钠/氢逆向转运蛋白基因Ha_ydjM。生物信息学分析表明,该基因属于YdjM超家族成员,是一个未知功能的膜蛋白,系统发育分析证实,其与来自Halobacillus sp.Marseille-P3789的YdjM(蛋白登录号WP_101846656.1)家族成员聚在一起但形成独立分支。研究发现,该基因能够恢复大肠杆菌突变株KNabc对0.2mol/LNaCl和5mmol/LLiCl的耐受特性,并且耐受碱性pH8.0。功能分析显示,该蛋白呈现pH依赖的钠/氢逆向转运蛋白活性,转运动力学分析表明,Na^+、K^+、Li^+在KNabc中Km值分别是0.43±0.05mmol/L、0.49±0.06mmol/L、0.64±0.06mmol/L,即对Na^+、K^+、Li^+的亲和力分别是Na^+>K^+>Li^+。综上所述,Ha_ydjM代表了一种新型的钠/氢逆向转运蛋白,这丰富了YdjM超家族成员,并为其他未知膜蛋白功能分析提供依据。

In prokaryotes,Na^+/H^+ antiporters catalyze the efflux of intracellular alkali cations,such as Na^+,Li^+ or K^+ in exchange for external protons,which play vital roles in reducing the cytoplasmic concentration of toxic alkali cations and supporting intracellular pH homeostasis under alkaline conditions.To obtain as many (especially novel) Na^+/H^+ antiporter genes as possible,genomic DNA from the Halobacillus Y5 was screened,then partially digested with Sau3AI selected in E.coli KNabc,which lacks three major Na^+/H^+ antiporters.The gene designated Ha_ydjM (ydjM from the Halobacillus Y5) was screened,which showed the noval Na^+/H^+ antiporter,belong to a member of YdjM superfamilly.It is a membrane protein of unknown function.Phylogenetic analysis based on the neighbour-joining algorithm showed that Ha-YdjM showed the closer phylogenetic relationship with YdjM(accession version No.WP_101846656.1) from Halobacillus sp.Marseille -P 3789 but it is a separate branch.The presence of Ha-YdjM conferred tolerance of E.coli KNabc to NaCl up to 0.2mol/L and to LiCl up to 5mmol/L and to an alkaline pH 8.0.pH-dependent Na^+/H^+ antiport activity was detected from everted membrane vesicles prepared from E.coli KNabc/Ha-YdjM but not those of KNabc/pUC18.The Michaelis-Menten kinetics analysis showed that Km values for Na^+,K^+,Li + were 0.43±0.05mmol/L、0.49±0.06mmol/L、0.64±0.06mmol/L,respectively,indicating that the preference of Ha-YdjM for the transported cations was Na^+ > K^+ > Li +.Taken together,Ha_ydjM represents a novel Na^+/H^+ antiport.This study found new members of the YdjM superfamily and provided insights for functions of other unknown membrane members.