tailieunhanh - Báo cáo khoa học về: Mitochondria regulate platelet metamorphosis induced by opsonized zymosan A – activation and long-term commitment to cell death

Changes in the mitochondrial membrane potential play a key role in deter-mining cell fate. Mitochondria membrane hyperpolarization has been found to occur after cell activation, . in lymphocytes, whereas depolar-ization is associated with apoptosis. | Accessory active site residues of Streptomyces sp. N174 chitosanase Variations on a common theme in the lysozyme superfamily 1 2 1r - .1 Marie-Eve Lacombe-Harvey Tamo Fukamizo Julie Gagnon Mariana G. Ghinet Nicole Dennhart3 Thomas Letzel3 and Ryszard Brzezinski1 1 Departement de Biologie Centre d Etude et de Valorisation de la Diversite Microbienne Universite de Sherbrooke Canada 2 Department of Advanced Bioscience Kinki University Nara Japan 3 Department for Basic Life Sciences Technische Universitat Munchen Freising-Weihenstephan Germany Keywords chitinase chitosanase glycoside hydrolase inverting mechanism lysozyme Correspondence R. Brzezinski Departement de Biologie Universite de Sherbrooke 2500 boul. de l Universite Sherbrooke QC J1K 2R1 Canada Fax 1 819 821 8049 Tel 1 819 821 8000 ext 61077 E-mail Received 22 September 2008 revised 26 November 2008 accepted 3 December 2008 doi The chitosanase from Streptomyces sp. N174 CsnN174 is an inverting glycoside hydrolase belonging to family 46. Previous studies identified Asp40 as the general base residue. Mutation of Asp40 into glycine revealed an unexpectedly high residual activity. D40G mutation did not affect the stereochemical mechanism of catalysis or the mode of interaction with substrate. To explain the D40G residual activity putative accessory catalytic residues were examined. Mutation of Glu36 was highly deleterious in a D40G background. Possibly the D40G mutation reconfigured the catalytic center in a way that allowed Glu36 to be positioned favorably to perform catalysis. Thr45 was also found to be essential. Thr45 is thought to orientate the nucleophilic water molecule in a position to attack the glycosidic link. The finding that expression of heterologous CsnN174 in Escherichia coli protects cells against the antimicrobial effect of chitosan allowed the selection of active chitosanase variants after saturation mutagenesis. Thr45 could be .