The structural basis of fungal glucuronoyl esterase activity on natural substrates

Caroline Mosbech,Anne S. Meyer,Heidi A. Ernst,Peter Westh,Sine Larsen,Annette E. Langkilde,Jane W. Agger

doi:10.1038/s41467-020-14833-9

Caroline Mosbech, Anne S. Meyer + Show 5 more

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https://doi.org/10.1038/s41467-020-14833-9

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Abstract

Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an α/β-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.

Highlights

Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds
That a two-domain glucuronoyl esterase from the basidiomycete Cerrena unicolor (CuGE) with high efficiency can catalyze the release of large soluble aldouronic acids from a lignin-rich birchwood fraction, most likely containing the already mentioned ester-linked LCCs5
These CuGE variants were recombinantly produced in Pichia pastoris and subsequently purified

Summary

Introduction

Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. It was recently discovered that an insoluble hardwood lignin-preparation (LRP) is amenable to enzymatic hydrolysis by a glucuronoyl esterase (GE, EC 3.1.1.B11) releasing aldouronic acids[5]. This opens for the opportunity to use glucuronoyl esterases as a benign tool for processing of lignin in selective removal of residual carbohydrates in contrast to the harsh physio/chemical extractions otherwise used. There is only one report of a CE15 complex, the StGE2:monosaccharide complex[12], this study is not supported by any functional data

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