Probing the Functions of Carbohydrate Binding Modules in the CBEL Protein from the Oomycete Phytophthora parasitica.

Michael O’Donohue,Laurent Heux,Hélène Texier,Gianluca Cioci,Bernard Dumas,Elodie Gaulin,Thomas Martinez,Claire Dumon,Claude Lafitte,Virginie Nahoum

doi:10.1371/journal.pone.0137481

Abstract

Oomycetes are microorganisms that are distantly related to true fungi and many members of this phylum are major plant pathogens. Oomycetes express proteins that are able to interact with plant cell wall polysaccharides, such as cellulose. This interaction is thought to be mediated by carbohydrate-binding modules that are classified into CBM family 1 in the CAZy database. In this study, the two CBMs (1–1 and 1–2) that form part of the cell wall glycoprotein, CBEL, from Phytophthora parasitica have been submitted to detailed characterization, first to better quantify their interaction with cellulose and second to determine whether these CBMs can be useful for biotechnological applications, such as biomass hydrolysis. A variety of biophysical techniques were used to study the interaction of the CBMs with various substrates and the data obtained indicate that CBEL’s CBM1-1 exhibits much greater cellulose binding ability than CBM1-2. Engineering of the family 11 xylanase from Talaromyces versatilis (TvXynB), an enzyme that naturally bears a fungal family 1 CBM, has produced two variants. The first one lacks its native CBM, whereas the second contains the CBEL CBM1-1. The study of these enzymes has revealed that wild type TvXynB binds to cellulose, via its CBM1, and that the substitution of its CBM by oomycetal CBM1-1 does not affect its activity on wheat straw. However, intriguingly the addition of CBEL during the hydrolysis of wheat straw actually potentiates the action of TvXynB variant lacking a CBM1. This suggests that the potentiating effect of CBM1-1 might not require the formation of a covalent linkage to TvXynB.

Highlights

Plant cell walls constitute the most abundant source of renewable carbon on Earth, the industrial extraction of their constituent sugars is difficult, because the polysaccharides are chemically complex, structured and interlinked, and are embedded in a matrix of lignin, which is a high molecular weight, amorphous polyphenolic polymer [1]
cellulose-binding elicitor lectin (CBEL) is a glycoprotein from Phytophthora parasitica that is composed of two CBM1s and two non-catalytic modules designated PAN/APPLE and are known to bind to crystalline cellulose in vitro (Fig 1)
A previous mutagenesis study has provided evidence that Y52 and Y188 located on the surface of CBM1-1 and CBM1-2 respectively are important for cellulose binding, since the double mutant CBELY52-Y188 was deprived of binding ability [27]

Summary

Introduction

Plant cell walls constitute the most abundant source of renewable carbon on Earth, the industrial extraction of their constituent sugars is difficult, because the polysaccharides are chemically complex, structured and interlinked, and are embedded in a matrix of lignin, which is a high molecular weight, amorphous polyphenolic polymer [1] To overcome these difficulties, microorganisms that degrade plant cell walls in natural ecosystems have developed a number of strategies, which include plant cell wall-specific enzymes and related proteins that together surmount the many natural obstacles that confer recalcitrance to plant cell wall [2]. This study suggests that the possession of a CBM would confer an advantage for polysaccharide degrading enzymes in the context of complex substrate (e.g. lignocellulosic biomass)

Methods

Results

Conclusion