Characterization of the effects of C-terminal pro-sequence on self-inactivation of Stereum purpureum endopolygalacturonase I
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 char...
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Published in | FEMS microbiology letters Vol. 362; no. 17; p. fnv134 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
England
Oxford University Press
01.09.2015
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1574-6968 0378-1097 1574-6968 |
DOI: | 10.1093/femsle/fnv134 |