Molecular cloning and functional analysis of pppg1 encoding endo-polygalacturonase in Phytophthora parasitica
Date Issued
2006
Date
2006
Author(s)
Yan, Hao-Zhi
DOI
en-US
Abstract
The deduced amino acid sequence of pppg1, which encodes a polygalacturonase in the oomycete plant pathogen Phytophthora parasitica, contains eleven putative N-glycosylation sites. To understand the significance of N-glycosylation on the enzymatic activity of PPPG1, site-directed mutagenesis was employed to generate a serial of single and multiple mutations on the N-glycosylation sites. Recombinant mutant proteins were expressed using a yeast (Pichia pastoris) protein expression system and their biochemical characteristics were analyzed. The results indicated that mutations in the N-glycosylation sites resulted in a significant loss of the endoPG activity compared to that of the wild-type protein, especially mutation at N8, suggesting the importance of N-glycosylation on the structure and thereby the enzymatic function of the PPPG1. Besides, mutation in positions N1, N3, N4, N10, and N11 caused a reduction in thermostability of the recombinant proteins. It is thus suggested that N-glycosylations on these amino acid residues might be involved in preventing the protein from the deleterious effect caused by heat. N-glycosylation is also important for the secretion of PPPG1. Furthermore, it was demonstrated that the replacement of the N-terminus of PPPG1 with that of PG3b, another gene encoding endopolygalacturonase in P. parasitica which contains no N-glycosylation site, resulted in restoration of activity to N1-4 mutated PPPG1, indicating that N-glycosylation in the first four N-glycosylation sites of pppg1 might play a major role in the formation of parallel β-helix. Nevertheless, the chimeric protein exhibited a reduction in thermostability, implicating the essential function of N-glycosylation in increasing the stability of the enzyme against thermal unfolding. In the meanwhile, mutations were generated in six amino acid residues which are highly conserved among endoPGs. Analysis of the biochemical properties of these mutants indicated that residues Asp209, Asp230, Asp231, H252, and K290 play very important roles in pectin hydrolysis.
Subjects
疫病菌
多聚半乳糖醛酸酶
Phytophthora parasitica
polygalacturonase
PG
Type
other