The plant cell wall is a complex structure which is mainly composed of polymers like cellulose, hemicellulose, pectin and lignin. Cellulose is a linear polymer of β-(1, 4)-D-glucose units and is broken down by enzymes such as cellulases and beta-glucosidases into oligosaccharides and/or monosaccharides. There are three main classes of cellulose-hydrolysing enzymes; Endoglucanases, Exoglucanases/Cellobiohydrolases and β-glucosidases [1]. Endoglucanases attack the amorphous regions of cellulose and make random cuts all along the cellulose chain, decreasing the viscosity rapidly and releasing oligosaccharides of varied lengths as the products [2]. On the other hand, exoglucanases prefer the crystalline regions of cellulose and act from the ends of the cellulose polymer in a processive manner releasing cellobiose as the product [2], [3]. Exoglucanases are further classified into two types, Type 1 and 2 exoglucanases. While Type 1 exoglucanases act from the reducing ends of the cellulose chain, Type 2 exoglucanases act from the non-reducing end [1]. Even though β-glucosidases are not canonical cellulases per se, they are a very important component of the cellulase degrading enzyme system as they convert cellobiose and short oligosaccharides into glucose molecules [4]. The synergistic action of all these three groups of enzymes results in a complete decomposition of the recalcitrant and complex cellulose molecule.

Plant cell wall degrading enzymes are important for the virulence of plant pathogens. Since cellulose is one of the major components of the plant cell walls, a majority of the phytopathogens secrete a battery of cellulose-hydrolysing enzymes. Xanthomonas oryzae pv. oryzae (Xoo), which is responsible for the bacterial blight disease in rice secretes a variety of cell wall degrading enzymes that include cellulases, xylanases and pectinases. Among the cellulases secreted by Xoo, cellobiosidase (CbsA) is shown to be an important virulence factor [5]. CbsA protein has an N-terminal catalytic domain which belongs to the glycosyl hydrolase-6 (GH6) family and a C-terminal fibronectin type III domain [6]. In our previous study, the crystal structure of the CbsA catalytic domain has been solved at 1.86 Å resolution [7]. The structure showed the presence of the characteristic N- and C-terminal surface loops enclosing the active site, thus confirming the catalytic domain of CbsA as an exoglucanase that cleaves the cellulose chain in a processive manner to release cellobiose units [7]. In the same study, we have also characterised an active site aspartate mutant (D131A) of CbsA which displayed endoglucanase activity.

Members of the GH6 family catalyse the breakage of the glycosidic bond with inversion of the configuration of the anomeric carbon [8]. In the proposed hydrolysis mechanism resulting in inversion, a deprotonated glutamate or aspartate residue functions as a catalytic base and abstracts a proton from catalytic water to facilitate the mounting of a nucleophilic attack on the anomeric carbon C1. Thus, the covalent bond between the anomeric carbon (C1) and oxygen is cleaved, concomitantly inverting the linkage from β-anomeric configuration to α-anomeric configuration. Meanwhile, the catalytic acid, mostly an aspartate, helps in the cleavage by donating its proton and promoting the departure of the oxygen, which is otherwise a poor leaving group [9].

There is a consensus on the identity of the catalytic acid involved in the hydrolysis based on the structural and biochemical studies on exoglucanases and endoglucanases (GH6 family) of both bacteria and fungi [10], [11], [12], [13], [14], [15], [16]. An active site aspartic acid is proposed to act as the catalytic acid in all these enzymes. However, the identity of the catalytic base has been a topic of debate. Two independent studies have proposed a novel mechanism, which involves a proton-transfer network instead of a single Bronsted base [15], [17].

In the present study, we report the mutation and biochemical characterisation of invariant residues of the CbsA catalytic domain, which are proposed to have a role in catalysis. We report that these mutations do not abolish the biochemical activity of CbsA but alter it in subtle ways. These mutations in the CbsA protein also affect Xoo virulence in rice.