A Novel Carbohydrate-binding Module from Sugar Cane Soil Metagenome Featuring Unique Structural and Carbohydrate Affinity Properties

Abstract

Carbohydrate-binding modules (CBMs) are appended to glycoside hydrolases and can contribute to the degradation of complex recalcitrant substrates such as the plant cell wall. For application in bioethanol production, novel enzymes with high catalytic activity against recalcitrant lignocellulosic material are being explored and developed. In this work, we report the functional and structural study of CBM_E1, which was discovered through a metagenomics approach and is the founding member of a novel CBM family, CBM81. CBM_E1, which is linked to an endoglucanase, displayed affinity for mixed linked β1,3-β1,4-glucans, xyloglucan, Avicel, and cellooligosaccharides. The crystal structure of CBM_E1 in complex with cellopentaose displayed a canonical β-sandwich fold comprising two β-sheets. The planar ligand binding site, observed in a parallel orientation with the β-strands, is a typical feature of type A CBMs, although the expected affinity for bacterial crystalline cellulose was not detected. Conversely, the binding to soluble glucans was enthalpically driven, which is typical of type B modules. These unique properties of CBM_E1 are at the interface between type A and type B CBMs.

Advances in molecular engineering of carbohydrate-binding modules

“…In all cases, substrate binding is mediated by aromatic amino acids, particularly tryptophan and tyrosine through hydrophobic and CH–π interactions, resulting in the formation of hydrophobic platforms at the CBM‐binding site. Hydrogen bonds formed by tyrosines or polar amino acids stabilize the interaction, although their contribution is more important for type B and C topologies …”

“…In general, type B CBMs cannot interact with planar substrates, such as crystalline cellulose, but can bind to a wide variety of glycans, such as xylans, mannans, galactans, and starch . Similar to type A CBMs, these interactions are governed by aromatic amino acids through hydrophobic and CH–π interactions.…”

“…Hydrogen bonds formed by tyrosines or polar amino acids stabilize the interaction, although their contribution is more important for type B and C topologies. 6,7 Because this type of topology mirrors that of the substrate, the main feature of type A CBMs is a planar and hydrophobic surface capable of binding insoluble carbohydrates, such as crystalline cellulose and chitin, through entropically driven CH-p interactions between the enzyme's aromatic amino acids and the sugar residues. It has been reported that type A CBMs present little or no affinity for oligosaccharides.…”

“…1 In general, type B CBMs cannot interact with planar substrates, such as crystalline cellulose, but can bind to a wide variety of glycans, such as xylans, mannans, galactans, and starch. 7,[9][10][11] Similar to type A CBMs, these interactions are governed by aromatic amino acids through hydrophobic and CH-p interactions. Hydrogen bonds established mainly by polar residues are determinants in the selectivity and affinity of CBMs for their substrates.…”

Revealing the insoluble metasecretome of lignocellulose-degrading microbial communities

“…Many of these proteins contain CBMs 31 but no catalytic domains identifiable from previously characterized proteins, suggesting that much of the metasecretome is yet to be understood.…”