Crystal structure and functional characterization of an oligosaccharide dehydrogenase from Pycnoporus cinnabarinus provides insights into fungal breakdown of lignocellulose

Abstract

Background Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin. Results In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a β(1→3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-π interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario. Conclusions Structure–function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.

Characterization of Fungal FAD-Dependent AA3_2 Glucose Oxidoreductases from Hitherto Unexplored Phylogenetic Clades

“…We performed a pair-wise sequence comparison between our four newly studied glucose oxidoreductases and structurally characterized members of the AA3_2 glucose oxidoreductases- An GOx (PDB entry 1CF3) of clade GOx I, a glucose dehydrogenase of clade GDH I from Aspergillus flavus ( Af GDH; PDB entry 4YNT) as well as a glucose dehydrogenase of clade GDH III from the basidiomycete P. cinnabarinus ( Pc GDHIII; PDB entry 6XUT). The structure of this latter enzyme was elucidated only recently, and because of the preferential activity of this enzyme toward oligosaccharides containing a β(1→3)-linked reducing glucose moiety such as laminaribiose or 1,3:1,4 β-glucotriose B (3 1 -β-D-cellobiosyl-glucose), it was termed oligosaccharide dehydrogenase [ 6 ]. For reasons of consistency with the nomenclature of the other AA3_2 members, we are here referring to this enzyme as Pc GDHIII.…”

“…The catalytic His pair is located in the proximity of the isoalloxazine ring, and the side chains of these histidines are stabilized by H-bonds with a Gln and a Glu. Most of the other residues forming the active-site cavity of Pc GDHIII are of aromatic or hydrophobic nature [ 6 ]. Active-site residues around the proposed substrate-binding site of Pc GDHIII and Rs GDHIII as well as Um GDHIII are well-conserved as is the catalytic His pair ( Figure 4 C,D).…”

“…Additionally, Tyr64 in Pc GDHIII stabilizes sugar binding through van der Waals interactions and a polar contact. These four residues are highly conserved in Rs GDHIII as well as Um GDHIII (only the position of Phe421 is substituted by a Tyr in Rs GDHIII), as they are typically also in other members of clade GDH III [ 6 ]. The funnel-shaped active site can accommodate two molecules of glucose, and it was proven that Pc GDHIII shows significantly higher catalytic efficiency for the disaccharide laminaribiose than for glucose, mainly because of a considerably more favorable Michaelis constant for the disaccharide.…”

“…It is striking though that two major clades of glucose oxidoreductases (GDH II and GDH III) are apparently comprising enzymes that are not primarily oxidizing glucose, and the name glucose oxidoreductase for this group of enzymes could be debated. Nevertheless, we are still referring to the name glucose oxidoreductases here, and did not adopt the term oligosaccharide dehydrogenases for the GDH III enzymes as had been suggested previously [ 6 ]. The reason for this is that all glucose oxidoreductases (GOx I-II and GDH I-III) share a close phylogenetic relation, and we want to avoid confusion with the CAZy auxiliary activity family 7, which is comprised of oligosaccharide oxidoreductases [ 29 ].…”

“…A good example for this latter type of enzymes is the glucose dehydrogenase from Pycnoporus cinnabarinus . Recently, it was shown that this enzyme shows much higher activity against laminaribiose (glucosyl-β(1→3)-glucose) than for glucose; in fact, it was suggested that this enzyme should be named as an oligosaccharide dehydrogenase [ 6 ].…”