tailieunhanh - Báo cáo khoa học: Identification and characterization of the metal ion-dependent L-alanoyl-D-glutamate peptidase encoded by bacteriophage T5

Although bacteriophage T5 is known to have lytic proteins for cell wall hydrolysis and phage progeny escape, their activities are still unknown. This is the first report on the cloning, expression and biochemical charac-terization of a bacteriophage T5 lytic hydrolase. | Identification and characterization of the metal ion-dependent L-alanoyl-D-glutamate peptidase encoded by bacteriophage T5 Galina V. Mikoulinskaia1 Irina V. Odinokova2 Andrei A. Zimin3 Valentina Ya. Lysanskaya3 Sergei A. Feofanov1 and Olga A. Stepnaya3 1 Branch of Shemyakin Ovchinnikov s Institute of Bioorganic Chemistry RAS Pushchino Moscow Russia 2 Institute of Theoreticaland ExperimentalBiophysics RAS Pushchino Moscow Russia 3 Skryabin s Institute of Biochemistry and Physiology of Microorganisms RAS Pushchino Moscow Russia Keywords bacteriophage T5 endolysin Gram-negative holin L-alanoyl-D-glutamate peptidase Correspondence G. V. Mikoulinskaia Branch of Shemyakin Ovchinnikov s Institute of Bioorganic Chemistry RAS Prospekt Nauki 6 Pushchino Moscow Region 142290 Russia Fax 7 4967 330527 Tel 7 4967 731780 E-mail mikulinskaya@ Received 14 August 2009 revised 14 October 2009 accepted 16 October 2009 doi Although bacteriophage T5 is known to have lytic proteins for cell wall hydrolysis and phage progeny escape their activities are still unknown. This is the first report on the cloning expression and biochemical characterization of a bacteriophage T5 lytic hydrolase. The endolysin-encoding lys gene of virulent coliphage T5 was cloned in Escherichia coli cells and an electrophoretically homogeneous product of this gene was obtained with a high yield 78 of total activity . The protein purified was shown to be an L-alanoyl-D-glutamate peptidase. The enzyme demonstrated maximal activity in diluted buffers 25-50 mm at pH . The enzyme was strongly inhibited by EDTA and BAPTA and fully reactivated by calcium manga-nese chlorides. It was found that along with E. coli peptidoglycan peptidase of bacteriophage T5 can lyse peptidoglycans of other Gram-negative microorganisms Pectobacterium carotovorum Pseudomonas putida Proteus vulgaris and Proteus mirabilis . This endolysin is the first example of an L-alanoyl-D-glutamate .