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

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.

Structural and molecular dynamics studies of a C1‐oxidizing lytic polysaccharide monooxygenase from Heterobasidion irregulare reveal amino acids important for substrate recognition

“…This finding was later confirmed upon the solution of the first LPMO‐substrate structure, wherein the Lentinus similis LPMO9A ( Ls LPMO9A) complexed with cello‐oligosaccharide ligands (cellotriose and cellohexose) confirmed that the substrates bound to the proposed surface region by both hydrophobic interactions and hydrogen bonds . Recently published crystal structures of Ls LPMO9A in complex with various types of hemicellulosic oligosaccharide ligands provided further molecular‐level evidence by which we can begin to understand the broad substrate specificity of AA9 LPMOs .…”

“…). Residues within these loop regions have previously been observed to interact with cellulosic substrates, as determined by either crystallographic or computational approaches . Compared to some of the C1‐specific LPMO structures ( Nc LPMO9F, Pc LPMO9D, and Tt LPMO9E), the L2 loop of Hi LPMO9B is extended with an insertion of 10 amino acids (Fig.…”

“…Lytic polysaccharide monooxygenases (LPMOs) are a class of copper‐dependent enzymes that use an oxidative mechanism to cleave natural polysaccharide substrates, such as cellulose, xylan, xyloglucan, glucomannan, mixed‐linkage β‐glucan, chitin, and starch . LPMO‐encoding genes have been discovered in the genomes of a wide range of microorganisms, including fungi and bacteria .…”

“…For LPMOs that specifically carry out oxidization at the C1 carbon of the pyranose ring (C1‐specific LPMOs), only oxidative cellulolytic activities have been reported . Activities against hemicellulosic substrates have also been reported but have mainly been found in LPMOs able to oxidize at the C4 position, including C4‐specific (oxidize at C4) or C1/C4‐active (oxidize at both C1 and C4) LPMOs .…”

“…However, elucidating LPMO–substrate interactions at the molecular level remains a significant challenge, as the most common AA9 LPMO substrate is insoluble cellulose . Accordingly, previous investigations of LPMO–substrate interactions have focused on LPMOs active on soluble polysaccharides . As determined by NMR spectroscopy, soluble β‐glucan substrates appeared to interact with the active site located on the flat surface of the C4‐specific Neurospora crassa Nc LPMO9C .…”