Crystal Structure and Computational Characterization of the Lytic Polysaccharide Monooxygenase GH61D from the Basidiomycota Fungus Phanerochaete chrysosporium

Miao Wu,Gregg T Beckham,Anna M Larsson,Takuya Ishida,Seonah Kim,Christina M Payne,Michael E Himmel,Michael F Crowley,Svein J Horn,Bjørge Westereng,Kiyohiko Igarashi,Masahiro Samejima,Jerry Ståhlberg,Vincent G.H Eijsink,Mats Sandgren

doi:10.1074/jbc.m113.459396

Abstract

Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain.

Highlights

Lytic polysaccharide monooxygenases (LPMOs) represent a recently discovered enzymatic route to cleave carbohydrates
We showed that PchGH61D is a copper-dependent LPMO with activity on Avicel, filter paper, and phosphoric acid-swollen cellulose, which oxidizes at the C1 carbon
Computational Study of PchGH61D-Cellulose Interactions— To conduct classical MD simulations of PchGH61D with a copper ion bound in the enzyme, the charge redistribution in the active center upon copper binding was examined with electronic structure calculations, as described in the supplemental material and shown in supplemental Fig. S2

Summary

Background

Lytic polysaccharide monooxygenases (LPMOs) represent a recently discovered enzymatic route to cleave carbohydrates. A new class of enzymes was discovered that uses copper-dependent oxidative pathways for the cleavage of glycosidic linkages [2,3,4,5] These oxidative enzymes, referred to here as lytic polysaccharide monooxygenases (LPMOs), have garnered significant interest because they enhance degradation of recalcitrant polysaccharides, such as chitin and cellulose, when added to GH mixtures [6, 7]. The flat catalytic binding surfaces of LPMOs are putatively suited to cleave glycosidic linkages without decrystallizing polymer chains [13, 22, 23], whereas endoglucanases, with a catalytic cleft, are thought to mainly act on more accessible, amorphous regions [24] This may explain why these two enzyme classes are synergistic [2]. This study contributes to the expanding repertoire of LPMO structures and identifies key interactions with the hydrophobic face of cellulose, which will aid in describing the mechanism and specificity of these important enzymes

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

CONCLUSIONS