X4 Modules Represent a New Family of Carbohydrate-binding Modules That Display Novel Properties

Abstract

The hydrolysis of the plant cell wall by microbial glycoside hydrolases and esterases is the primary mechanism by which stored organic carbon is utilized in the biosphere, and thus these enzymes are of considerable biological and industrial importance. Plant cell walldegrading enzymes in general display a modular architecture comprising catalytic and non-catalytic modules. The X4 modules in glycoside hydrolases represent a large family of non-catalytic modules whose function is unknown. Here we show that the X4 modules from a Cellvibrio japonicus mannanase (Man5C) and arabinofuranosidase (Abf62A) bind to polysaccharides, and thus these proteins comprise a new family of carbohydratebinding modules (CBMs), designated CBM35. The Man5C-CBM35 binds to galactomannan, insoluble amorphous mannan, glucomannan, and manno-oligosaccharides but does not interact with crystalline mannan, cellulose, cello-oligosaccharides, or other polysaccharides derived from the plant cell wall. Man5C-CBM35 also potentiates mannanase activity against insoluble amorphous mannan. Abf62A-CBM35 interacts with unsubstituted oat-spelt xylan but not substituted forms of the hemicellulose or xylo-oligosaccharides, and requires calcium for binding. This is in sharp contrast to other xylan-binding CBMs, which interact in a calcium-independent manner with both xylo-oligosaccharides and decorated xylans.

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…Many CBMs are metalloproteins; however, the role of metal ions in CBM-ligand interactions has only recently been discovered. Xylan recognition by the family 35 CBM of the Cellvibrio japonicus enzyme Abf62A is calcium-dependent; however, the structure of this module is unknown [72]. More recently, the recognition of xylooligosaccharides by the family 36 CBM from the Pa. polymyxa enzyme Xyn43A was also demonstrated to be calcium-dependent [44a].…”

Differential recognition of plant cell walls by microbial xylan-specific carbohydrate-binding modules

“…We have selected six CBMs, located in families 2b, 4, 6, 15, 22, and 35, listed in Table 1, which display specificity for purified xylans (6,10,(14)(15)(16)(17). The data presented in this study show that there is significant variation in the specificity of the different CBMs for plant cell walls, indicating that the structure and͞or context of the xylans embedded in these composite structures is a critical specificity determinant for these protein modules.…”

“…Although CBM6, CBM4-2, CBM15, and CBM22-2 bind to both unsubstituted xylan and arabinoxylan, CBM35 displays a strong preference for unsubstituted xylan polymers, compared to MGX and RAX (10) but, in addition, shows significant recognition of GAX (the likely target ligand of this module in primary cell walls, see above). Therefore, it is likely that the decorations in GAX are either relatively sparse compared to RAX and MGX, or the substitutions are present in discrete blocks thus presenting significant regions of undecorated xylan, although it is also possible that CBM35 recognizes the specific substitutions in GAX.…”

“…In addition, this module is unique in displaying no affinity for xylohexaose. The actual affinity of the six CBMs for purified xylans (Table 2) reflects their microbial origin; proteins from thermophilic bacteria bind more tightly to their target ligands (CBM4-2, CBM6, and CBM22-2) (6, 16, 17) than individual CBM2b modules, CBM15 or CBM35, which are derived from mesophilic prokaryotes (10,15,17). The relatively high affinity displayed by CBM2b-1-2 is due to the avidity effect of joining two CBMs together that bind to polysaccharides, which contain multiple protein binding sites (15,20).…”

“…Although the ligand specificity of type A CBMs is highly conserved, the sugar polymers recognized by different members of type B families can be very diverse. For example, the CBM4 and CBM6 families both contain members that recognize xylan, ␤-1,4-glucan and ␤-1,3-glucan (5-8), whereas the CBM35 family contains proteins that, currently, have been shown to bind to xylan and mannan (9,10).…”