Characterization of the effects of C-terminal pro-sequence on self-inactivation of Stereum purpureum endopolygalacturonase I

• Published in: FEMS microbiology letters Vol. 362; no. 17; p. fnv134
• Main Authors: Hamada, Shigeki, Toda, Kensuke, Ogawa, Sayaka, Kubota, Keisuke, Miyairi, Kazuo
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.09.2015
• Subjects: Agaricales - enzymology; Agaricales - genetics; Alanine; Amino Acid Sequence; Deactivation; Endopolygalacturonase; Enzyme Precursors - chemistry; Enzyme Precursors - metabolism; Glutamates; Hydrogen-Ion Concentration; Inactivation; Kinetics; Malus - microbiology; Microbiology; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Pectins - metabolism; pH effects; Plant diseases; Polygalacturonase - chemistry; Polygalacturonase - genetics; Polygalacturonase - metabolism; Sequence Homology, Amino Acid; Serine Endopeptidases - metabolism; Silver-leaf disease; Stereum purpureum; Substrate preferences; Substrates; site-direct mutagenesis; endopolygalacturonase; kinetics; pro-sequence; inactivation; pectin; Stereum purpureum
• ISSN: 1574-6968; 0378-1097; 1574-6968
• DOI: 10.1093/femsle/fnv134

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. This paper describes the analysis of an unknown suppression mechanism of endopolygalacturonase activity using detailed enzyme characterizations and site-directed mutagenesis.