A Novel Carbohydrate-binding Protein is a Component of the Plant Cell Wall-degrading Complex of Piromyces Equi

The recycling of photosynthetically fixed carbon by the action of microbial plant cell wall hydrolases is a fundamental biological process that is integral to one of the major geochemical cycles and, in addition, has considerable industrial potential. Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Anaerobic fungi that colonize herbivores are the most efficient plant cell wall degraders known, and this activity is vested in a high molecular weight complex that binds tightly to the plant cell wall. To investigate whether plant cell wall attachment is mediated by noncatalytic proteins, a cDNA library of the anaerobic fungus Piromyces equi was screened for sequences that encode noncatalytic proteins that are components of the cellulase-hemicellulase complex. A 1.6-kilobase cDNA was isolated encoding a protein of 479 amino acids with a M(r) of 52548 designated NCP1. The mature protein had a modular architecture comprising three copies of the noncatalytic dockerin module that targets anaerobic fungal proteins to the cellulase-hemicellulase complex. The two C-terminal modules of NCP1, CBM29-1 and CBM29-2, respectively, exhibit 33% sequence identity with each other but have no homologues in protein data bases. A truncated form of NCP1 comprising CBM29-1 and CBM29-2 (CBM29-1-2) and each of the two individual copies of CBM29 bind primarily to mannan, cellulose, and glucomannan, displaying the highest affinity for the latter polysaccharide. CBM29-1-2 exhibits 4-45-fold higher affinity than either CBM29-1 or CBM29-2 for the various ligands, indicating that the two modules, when covalently linked, act in synergy to bind to an array of different polysaccharides. This paper provides the first report of a CBM-containing protein from an anaerobic fungal cellulase-hemicellulase complex. The two CBMs constitute a novel CBM family designated CBM29 whose members exhibit unusually wide ligand specificity. We propose, therefore, that NCP1 plays a role in sequestering the fungal enzyme complex onto the plant cell wall.