“…5 ; Supplementary Table 3 ). Like the Ls AA9A:Cell 6 structure 33 , the main interactions to Cell 5 are a network of hydrogen bonds by Asn28, His66 and Asn67 interacting with O2 and O3 at subsite +2, and the interaction with MeHis1 at subsite +1 38 . Fig.…”
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.
“…5 ; Supplementary Table 3 ). Like the Ls AA9A:Cell 6 structure 33 , the main interactions to Cell 5 are a network of hydrogen bonds by Asn28, His66 and Asn67 interacting with O2 and O3 at subsite +2, and the interaction with MeHis1 at subsite +1 38 . Fig.…”
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.
“…The structures revealed polar residues in a loop denoted L3 (after notation in (25)) interacting with cellooligosaccharides near the active site and demonstrated for the first time that a conserved Tyr (Y203 in LsAA9A) located on the same surface was involved in substrate binding (22,23). In addition these structures revealed a new and highly unusual lone pair … π aromatic interaction between the pyranose ring O5 and the imidazole of the N-terminal His of the His brace, further highlighting its importance for LPMO function (29).…”
mentioning
confidence: 90%
“…Since the bioinformatics analysis and transcriptomic data suggested that Arg-AA9s might be functionally expressed during fungal growth on biomass, we sought to structurally and functionally characterise one of these proteins. The Polyporales fungus L. similis contains at least seven genes encoding proteins that are classified as AA9 of which LsAA9A has been well characterized (22,23,29). Also among these proteins is a single domain Arg-AA9 protein of 221 amino acids (aa) residues which we denote LsAA9B (GH61-5 in (32), GenBank accession MN265867).…”
Section: Lsaa9b An Aa9 With An N-terminal Arg From Lentinus Similismentioning
confidence: 99%
“…This surface can have varying contour and polarity, presumably dictating substrate specificity (19,23,25,26). Additionally, for some AA9 LPMOs activity has been demonstrated on soluble β-1,4-linked polysaccharides and also oligosaccharides (17,22,23,27,28), a feature which was instrumental in the determination of the first enzyme-substrate complex structures of an AA9 from Lentinus similis (LsAA9A) (22,29). The structures revealed polar residues in a loop denoted L3 (after notation in (25)) interacting with cellooligosaccharides near the active site and demonstrated for the first time that a conserved Tyr (Y203 in LsAA9A) located on the same surface was involved in substrate binding (22,23).…”
Running title: An unusual AA9 naturally lacking the His-brace motif *leila@chem.ku.dk, Universitetsparken 5, 2100 Copenhagen, DK (Phone: +45 35 32 02 95; Fax: +45 35 32 03 22). § Current Address.
AbstractLytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family, display a natural N-terminal His to Arg substitution (Arg-AA9). These are found almost entirely in the phylogenetic fungal class Agaricomycetes, associated with wood-decay, but no function has been demonstrated for any Arg-AA9.Through bioinformatics, transcriptomic and proteomic analyses we present data, which suggest that Arg-AA9 proteins could have a hitherto unidentified role in fungal degradation of lignocellulosic biomass in conjunction with other secreted fungal enzymes. We present the first structure of an Arg-AA9, LsAA9B, a
“…The ability to cleave polymers is enabled by a characteristic, flat binding-site consisting of aromatic and hydrophilic amino acids for the interaction with sugar moieties. An exposed active-site copper is held in place by a conserved, "histidine brace" motif [11][12][13]. These structural features enable LPMOs to attack polysaccharide surfaces inaccessible to hydrolases [13,14].…”
Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from Neurospora crassa during catalysis. LPMOs require a reduction in the active-site copper for catalytic activity. We show that copper reduction in NcLPMO9C leads to structural rearrangements and compaction around the active site. However, longer exposure to the reducing agent ascorbic acid also initiated an uncoupling reaction of the bound oxygen species, leading to oxidative damage, partial unfolding, and even fragmentation of NcLPMO9C. Interestingly, no changes in the hydrogen/deuterium exchange rate were detected upon incubation of oxidized or reduced LPMO with crystalline cellulose, indicating that the LPMO-substrate interactions are mainly side-chain mediated and neither affect intraprotein hydrogen bonding nor induce significant shielding of the protein surface. On the other hand, we observed a protective effect of the substrate, which slowed down the autooxidative damage induced by the uncoupling reaction. These observations further complement the picture of structural changes during LPMO catalysis.
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