Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module

Abstract

A novel ␤-(1,3)-glucanase gene designated lamC, cloned from Corallococcus sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exoacting enzyme on ␤-(1,3)(1,3)-and ␤-(1,6)-linked glucan substrates and an endoacting enzyme on ␤-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward ␤-(1,3)(1,3)-linked glucans. The carbohydratebinding assay showed that the fascin-like module was a novel ␤-(1,3)-linked glucanbinding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules. -(1,3)-glucan, which is the main cell wall component in yeast and filamentous fungi and a structural polysaccharide (e.g., callose) in plants and is also found in exopolysaccharides produced by some bacteria (1). Based on their amino acid sequence similarity and secondary structure, ␤-(1,3)-glucanases are classified mainly into glycoside hydrolase family 16 (GH16) and GH17. However, these two families have the same hydrolytic mechanism with anomeric retention (2).Numerous genes encoding ␤-(1,3)-glucanase have been cloned and characterized from different sources, including varieties of plants (3-5), bacteria, and archaea, such as Bacillus circulans (6), Paenibacillus (7, 8), Thermotoga neapolitana (1), Rhodothermus marinus (9), and Pyrococcus furiosus (10). Few glucanases exhibit broad substrate linkage specificity, although Lafond et al. cloned a gene from Podospora anserina that encodes a broad-specificity ␤-glucanase acting on ␤-(1,3)-, ␤-(1,4)-, and ␤-(1,6)-glucans (11).Many polysaccharide-degrading enzymes display a modular structure, in which a catalytic module is attached to one or more noncatalytic modules (8, 12, 13). The impact of the noncatalytic modules on the enzymatic properties of ␤-(1,3)-glucanase

A novel outer membrane β-1,6-glucanase is deployed in the predation of fungi by myxobacteria

“…This hypothesis is supported by the observation that, in other genera of myxobacteria, homologs of GluM are widely distributed (Table S7), which is in contrast to the rare occurrence of β-1,3-glucanases in myxobacteria (Table S10). No β-1,3-glucanase activity was detected from the supernatant of strain EGB, and only negligible enzymatic and antifungal β-1,3-glucanase activities were previously reported in strain EGB [34]. We predict that the impact of fungi on myxobacterial evolution led to occupation of a specific ecological niche by myxobacteria using a OM β-1,6-glucanase as a targeted weapon against fungi.…”

“…General properties of GluM, including optimal pH and temperature, were investigated using yeast glucan as the substrate. For determination of the hydrolysis pattern, GluM was incubated with 10 mg/mL yeast glucan, laminarin, pustulan and extracted ASDN faction (Methods in supplementary materials) at 30°C for 10 h, after which the soluble carbohydrate composition was analyzed by TLC on silica gel 60 plates (Merck, Germany) using n-butanol-methanol-H 2 O (8:4:3, v/v/v) as the solvent system [34]. The reaction products were visualized by spraying a sulfuric acid-methanol (1:1, v/v) solution onto the plate, followed by baking at 95°C for 10 min.…”

A GH13 glycoside phosphorylase with unknown substrate specificity from Corallococcus coralloides

“…Bacteriolytic myxobacteria can utilize several polysaccharides and maltotriose as carbon source, but mono-or disaccharides do not stimulate growth [45]. Several new glycoside hydrolases from Corallococcus species, including some from family GH13, have already been characterised in the past few years [46][47][48][49][50][51]. Those GH13 hydrolases indeed process maltooligosaccharides, but the GH13 phosphorylase discussed in this work did not appear to show such activity.…”

Whole-Genome Sequencing of Corallococcus sp. Strain EGB Reveals the Genetic Determinants Linking Taxonomy and Predatory Behavior

“…A β-(1,3)-glucanase (lamC) derived from Corallococcus sp. strain EGB, displays exo-mode activity toward β- (1,3)/ (1,6)-linked glucan substrates and endo-mode activity on β- (1,4)-linked glucan and xylan substrates, and shows lytic and antifungal activity against the plant pathogen Magnaporthe oryzae [11,12]. Another chitin hydrolase CcCti1 is identified from Corallococcus sp.…”