Differential Effect of Site-directed Mutations in pelC on Pectate Lyase Activity, Plant Tissue Maceration, and Elicitor Activity

Nobuhiro Kita,Carol M Boyd,Michael R Garrett,Frances Jurnak,Noel T Keen

doi:10.1074/jbc.271.43.26529

Nobuhiro Kita, Carol M Boyd + Show 3 more

Open Access

https://doi.org/10.1074/jbc.271.43.26529

Copy DOI

Abstract

Oligonucleotide site-directed mutations were introduced into the pelC gene of Erwinia chrysanthemi EC16 that directed single or double amino acid changes affecting disulfide linkages, calcium binding, catalysis, and protein folding. Subsequent characterization of the purified PelC mutant proteins demonstrated that pectinolytic function involves amino acids located near the calcium binding site rather than those surrounding an invariant vWiDH sequence. Wild-type PelC and the tested mutant proteins generally macerated plant tissue in proportion to their specific pectinolytic activity in vitro. However, some mutants gave higher maceration activity in plant tissue and elicited greater production of the phytoalexin, glyceollin, in soybean cotyledons than predicted by their in vitro pectinolytic activity. Most notable in this regard were three different mutations at lysine 172 with greatly reduced pectinolytic activity but as much elicitor activity as the wild-type protein. PelE macerated plant tissue 10 times more efficiently than PelC, as observed previously, but surprisingly showed equal activity in the elicitor assay. The results indicate that factors other than pectinolytic activity per se are involved in plant tissue maceration and elicitor activity.

Highlights

Icate their entrance into the Erwinia Out secretion system
PelC Mutants Constructed by Site-directed Mutagenesis— Screening of the PelC PCR mutant constructs by DNA sequencing at the mutation sites showed that the desired mutations were obtained from approximately 50% of the constructs analyzed, as theoretically expected
Overexpression of pelC Mutants—pelC and the various mutated genes in pRSET5A generally directed the production of large amounts of protein in E. coli cells, much of which was secreted into the periplasmic space (Fig. 2)

Summary

Introduction

Icate their entrance into the Erwinia Out secretion system. Because Escherichia coli cells lack the Out system, Pel proteins are not appreciably secreted extracellularly and instead accumulate in the periplasmic space. Structurebased multiple alignment of these Pel-related proteins revealed the presence of potentially catalytic, invariant amino acid residues, around the Ca2ϩ binding site and in the midregion of the core structure of the parallel ␤ helix, designated as the vWiDH region [25, 26] (see Fig. 1). These observations suggested that all of the Pel-related proteins might have pectinolytic activity. In this paper we report the construction of several oligonucleotide site-directed PelC mutant proteins and their characterization

Methods

Results

Conclusion