Structural and electronic determinants of lytic polysaccharide monooxygenase reactivity on polysaccharide substrates

T J Simmons,T Tryfona,M Tovborg,K S Johansen,L Ciano,T Tandrup,J C Poulsen,N Lenfant,L F L Wilson,P Dupree,P H Walton,B Henrissat,L Lo Leggio,K Schnorr,K E H Frandsen

doi:10.1038/s41467-017-01247-3

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are industrially important copper-dependent enzymes that oxidatively cleave polysaccharides. Here we present a functional and structural characterization of two closely related AA9-family LPMOs from Lentinus similis (LsAA9A) and Collariella virescens (CvAA9A). LsAA9A and CvAA9A cleave a range of polysaccharides, including cellulose, xyloglucan, mixed-linkage glucan and glucomannan. LsAA9A additionally cleaves isolated xylan substrates. The structures of CvAA9A and of LsAA9A bound to cellulosic and non-cellulosic oligosaccharides provide insight into the molecular determinants of their specificity. Spectroscopic measurements reveal differences in copper co-ordination upon the binding of xylan and glucans. LsAA9A activity is less sensitive to the reducing agent potential when cleaving xylan, suggesting that distinct catalytic mechanisms exist for xylan and glucan cleavage. Overall, these data show that AA9 LPMOs can display different apparent substrate specificities dependent upon both productive protein–carbohydrate interactions across a binding surface and also electronic considerations at the copper active site.

Highlights

Lytic polysaccharide monooxygenases (LPMOs) are industrially important copper-dependent enzymes that oxidatively cleave polysaccharides
To help understand the basis of AA9 substrate specificity, we searched for enzymes related to Lentinus similis AA9A (LsAA9A) that might cleave soluble oligosaccharides
From the LsAA9A clade, we identified an LPMO from Collariella virescens (CvAA9A; 46% sequence similarity to LsAA9A) that lacks some residues observed by Frandsen et al.[33] as being involved in enzyme–substrate interactions (Supplementary Fig. 1)

Summary

Introduction

Lytic polysaccharide monooxygenases (LPMOs) are industrially important copper-dependent enzymes that oxidatively cleave polysaccharides. LsAA9A activity is less sensitive to the reducing agent potential when cleaving xylan, suggesting that distinct catalytic mechanisms exist for xylan and glucan cleavage. Overall, these data show that AA9 LPMOs can display different apparent substrate specificities dependent upon both productive protein–carbohydrate interactions across a binding surface and electronic considerations at the copper active site. Recent ITC, NMR and docking studies of an AA9 LPMO from Neurospora crassa in contact with oligosaccharides revealed that more extended substrates had significantly higher binding affinities This is in accord with a multi-point interaction of the substrate with the LPMO surface where the surface loops in some LPMOs remote from the active site enhance binding affinity[21]. The +2 glucosyl residue exhibits a set of well-defined hydrogen-bonding interactions with amino-acid side chains (Asn[28], His[66] and Asn67) essentially locking this residue into a fixed position with respect to the active site

Methods

Results

Conclusion