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

Bruna Medeia Campos,Marcelo Vizona Liberato,Thabata Maria Alvarez,Letícia Maria Zanphorlin,Gabriela Cristina Ematsu,Hernane Barud,Igor Polikarpov,Roberto Ruller,Harry J Gilbert,Ana Carolina De Mattos Zeri,Fabio Marcio Squina

doi:10.1074/jbc.m116.744383

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.

Highlights

Carbohydrate-binding modules (CBMs) are capable of binding to different carbohydrates [5]
CBM_E1 Is the C-terminal Domain of CelE1, an Endoglucanase Derived from Sugar Cane Soil Metagenome—Previous functional screening of a metagenomic library using carboxymethyl cellulose as substrate identified a clone encoding a 427amino acid polypeptide [4]
The full-length CelE1 contains a putative N-terminal signal peptide [8], a catalytic module belonging to glycoside hydrolase family 5 (GH5), predicted on dbCAN web server [9], a serine-rich linker sequence, and a C-terminal region of unknown function, which was defined here as CBM_E1 (Fig. 1; accession number KJ917170)

Summary

Introduction

CBMs are capable of binding to different carbohydrates [5]. non-catalytic, CBMs can increase the enzymatic efficiency of its associated catalytic module against insoluble substrates through proximity effects [6]. It is proposed that CBM classification should be based on the structure and binding specificity of these modules: “surfacebinding to crystalline ligands” or type A; “endo-single chain glycan binding” or type B; and “exo-binding” or type C [1]. Type A CBMs bind to insoluble crystalline carbohydrates, crystalline cellulose, and/or chitin and have a planar binding site, usually composed of three aromatic amino acids, displaying little or no affinity for soluble carbohydrates. Type B CBMs, on the other hand, have a binding site shaped as a cleft and bind to a wide variety of glycans such as xylans, mannans, galactans, glucans of mixed linkage, and non-crystalline cellulose. The binding sites of type C CBMs display a pocket topology explaining why they recognize the non-reducing termini of glycans. CBM_E1 displays unique properties that are at the interface between type A and type B CBMs

Methods

Results

Conclusion