title: Characterization of the effects of C-terminal pro-sequence on self-inactivation of Stereum purpureum endopolygalacturonase I.
abstract: Endpolygalacturonase I from Stereum purpureum has been identified as a causative substance for the silver-leaf disease in apples. It possesses a unique pro-sequence in the C-terminal region that lacks endpolygalacturonases from any other origin. In this study, we analyzed and compared enzymatic characteristics between pro-form (pro-endoPG I) and mature form processed by V8 protease (endoPG I) and described the suppression activity of the pro-sequence. Of note, the optimal pH for pro-endoPG I activity shifted to pH 4.0 from pH 4.5-5.0 of endoPG I. The kinetic parameters indicated that the activity inhibition resulted from a pH-independent decrease of substrate affinity and pH-dependent deterioration of velocity by the pro-sequence. Analysis of site-directed mutations within pro-endoPG I showed that its α-helical structure includes two glutamates (E364 and E366) and alanine (A365), and its orientation by prolines (especially P348) in the pro-sequence played a significant role in its suppression activity. As for mutations in the mature domain, a marked reduction of suppression was observed for enzymes with mutations in H150, R220 and K253, indicating that the pro-sequence interacts with the active cleft by a few ionic bonds.
authors: Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan shamada@hirosaki-u.ac.jp.
authors: Shigeki
authors: S
authors: Hamada
authors: Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan.
authors: Kensuke
authors: K
authors: Toda
authors: Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan.
authors: Sayaka
authors: S
authors: Ogawa
authors: Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan.
authors: Keisuke
authors: K
authors: Kubota
authors: Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan.
authors: Kazuo
authors: K
authors: Miyairi
mesh_terms: D000363:Agaricales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D027845:Malus / Q000382:microbiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010368:Pectins / Q000378:metabolism; D011096:Polygalacturonase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism
chemical_list: D004792:Enzyme Precursors; D010368:Pectins; D011096:Polygalacturonase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase
keywords: Stereum purpureum; endopolygalacturonase; inactivation; kinetics; pectin; pro-sequence; site-direct mutagenesis