Elucidation of the molecular structure of OprD porin bearing the protease activity and the discovery of theporin homologous with OprD

近年来出现的多药耐药病原菌将对人类健康构成威胁。铜绿假单胞菌是一种对多种抗生素具有耐药性的条件致病菌，这种多药耐药被认为是由于外膜屏障和药物挤压系统的共同作用。我们发现铜绿假单胞菌外膜的孔蛋白形成小通道，再次形成抗生素穿透外膜的更高屏障。此外，我们还发现铜绿假单胞菌的一种孔蛋白OprD具有蛋白酶活性。在这个项目期间，进行了以下研究。(1)通过对比OprD与丝氨酸蛋白酶的氨基酸序列，推测OprD的催化残基可能是his56、Asp2O8和Ser296残基。为了鉴定催化残基，我们分别用其他氨基酸代替了这些氨基酸。这些突变蛋白的蛋白酶活性降低到天然OprD的0.1%。(2)当用Gin替换除上述氨基酸残基外的其他氨基酸残基（His367）时，发现该突变蛋白具有与天然OprD相似的活性。这些结果清楚地表明，QprD孔蛋白是一种具有蛋白酶活性的多功能蛋白。(3)抗OprD单克隆抗体与一个分子量为43 kDa的外膜蛋白具有交叉活性。该蛋白为OprE3。(4)对OprE3蛋白进行纯化，重组为脂质体，脂质体溶胀法检测，该蛋白为孔蛋白。(5)克隆了OprE3基因，命名为opr Q基因，并确定了该基因的核苷酸序列。将推断的OprE3氨基酸序列与OprD进行比较，发现OprE3与OprD高度同源。(6)为了用X射线衍射方法阐明OprD蛋白的三维结构，我们进行了OprD的纯化和蛋白结晶。我们尝试在各种条件下培养蛋白质晶体，并成功地培养了晶体。但由于晶体尺寸较小，不适合进行x射线衍射分析。我们现在正试图找出生长大而有序的OprD蛋白晶体的最佳条件。少

The recent emergence of the multidrug-resistant pathogens is going to becme a threaten for the human health. Pseudomos aeruginosa, an opportunistic pathogen possesses the resistance to a wide variety of antibiotics and this multidrug resistance is considered to be due to the concerted effects of the outer membrane barrier and the drug extrusion system.We showed that the porins in the outer membrane of P. aeruginosa form the small channels forming the higher barrier again the penetration of antibiotics through the outer membrane. Furthermore we revealed that OprD, one of porins in P.aeruginosa bears the protease activity. During the term of this project, the following studies were carried out.(1) The comparison of the amino acid sequences between OprD and serine protease lead the assumption that the catalytic residues of OprD may be Hisl56, Asp2O8 and Ser296 residues. In order to identify the catalytic residues, these amino acid were replaced with other amino acids, respectively, by usi … More ng the site-directed mutagenesis. The protease activity of these mutant proteins lowered to be 0.1% of that of native OprD.(2) When other amino acid residue (His367) besides the above ones was replaced with Gin, this mutant protein was found to eThit the similar activity with the native OprD.These results indicate clearly that QprD porin is a multi functional protein with protease activity.(3) The monoclonal antibodies against OprD showed the cross-activitywith one of the outer membrane proteins with the molecular weight of 43 kDa. This protein was revealed to be OprE3.(4) When OprE3 protein was purified, reconstituted into the liposome and ecamined by the liposome swelling method, this protein was showed to be a porin.(5) We cloned the OprE3 gene, which was denignated as opr Q gene, and determined the nucleotide sequence of this gene. When the deduced amino acid sequence of OprE3 was compared with that of OprD, OprE3 was revealed to be highly homologous with OprD.(6) In order to elucidate the three dimensinal structure of OprD protein by X ray diffraction method, we carried out the purification of OprD and protein crystallization. We have tried to grow the protein crystals under various conditions, and succeeded in growing the crystals. However, such crystals were not suitable for the x ray diffraction analysis since the crystal size was small. We are now trying to find out the best conditions for growing the large and well-ordered OprD protein crystals. Less