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

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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