Functional characterization of a lytic polysaccharide monooxygenase from Schizophyllum commune that degrades non-crystalline substrates.

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that use O2 or H2O2 to oxidatively cleave glycosidic bonds. LPMOs are prevalent in nature, and the functional variation among these enzymes is a topic of great interest. We present the functional characterization of one of the 22 putative AA9-type LPMOs from the fungus Schizophyllum commune, ScLPMO9A. The enzyme, expressed in Escherichia coli, showed C4-oxidative cleavage of amorphous cellulose and soluble cello-oligosaccharides. Activity on xyloglucan, mixed-linkage ß-glucan, and glucomannan was also observed, and product profiles differed compared to the well-studied C4-oxidizing NcLPMO9C from Neurospora crassa. While NcLPMO9C is also active on more crystalline forms of cellulose, ScLPMO9A is not. Differences between the two enzymes were also revealed by nuclear magnetic resonance (NMR) titration studies showing that, in contrast to NcLPMO9C, ScLPMO9A has higher affinity for linear substrates compared to branched substrates. Studies of H2O2-fueled degradation of amorphous cellulose showed that ScLPMO9A catalyzes a fast and specific peroxygenase reaction that is at least two orders of magnitude faster than the apparent monooxygenase reaction. Together, these results show that ScLPMO9A is an efficient LPMO with a broad substrate range, which, rather than acting on cellulose, has evolved to act on amorphous and soluble glucans.

Following the Fate of Lytic Polysaccharide Monooxygenases under Oxidative Conditions by NMR Spectroscopy.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of polysaccharides, such as cellulose and chitin. LPMO catalysis requires a reductant, such as ascorbic acid, and hydrogen peroxide, which can be generated in situ in the presence of molecular oxygen and various electron donors. While it is known that reduced LPMOs are prone to autocatalytic oxidative damage due to off-pathway reactions with the oxygen co-substrate, little is known about the structural consequences of such damage. Here, we present atomic-level insights into how the structure of the chitin-active SmLPMO10A is affected by oxidative damage using NMR and circular dichroism spectroscopy. Incubation with ascorbic acid could lead to rearrangements of aromatic residues, followed by more profound structural changes near the copper-active site and loss of activity. Longer incubation times induced changes in larger parts of the structure, indicative of progressing oxidative damage. Incubation with ascorbic acid in the presence of chitin led to similar changes in the observable (i.e., not substrate-bound) fraction of the enzyme. Upon subsequent addition of H2O2, which drastically speeds up chitin hydrolysis, NMR signals corresponding to seemingly intact SmLPMO10A reappeared, indicating dissociation of catalytically competent LPMO. Activity assays confirmed that SmLPMO10A retained catalytic activity when pre-incubated with chitin before being subjected to conditions that induce oxidative damage. Overall, this study provides structural insights into the process of oxidative damage of SmLPMO10A and demonstrates the protective effect of the substrate.

The impact of reductants on the catalytic efficiency of a lytic polysaccharide monooxygenase and the special role of dehydroascorbic acid.

Monocopper lytic polysaccharide monooxygenases (LPMOs) catalyse oxidative cleavage of glycosidic bonds in a reductant-dependent reaction. Recent studies indicate that LPMOs, rather than being O2 -dependent monooxygenases, are H2 O2 -dependent peroxygenases. Here, we describe SscLPMO10B, a novel LPMO from the phytopathogenic bacterium Streptomyces scabies and address links between this enzyme's catalytic rate and in situ hydrogen peroxide production in the presence of ascorbic acid, gallic acid and l-cysteine. Studies of Avicel degradation showed a clear correlation between the catalytic rate of SscLPMO10B and the rate of H2 O2 generation in the reaction mixture. We also assessed the impact of oxidised ascorbic acid, dehydroascorbic acid (DHA), on LPMO activity, since DHA, which is not considered a reductant, was recently reported to drive LPMO reactions. Kinetic studies, combined with NMR analysis, showed that DHA is unstable and converts into multiple derivatives, some of which are redox active and can fuel the LPMO reaction by reducing the active site copper and promoting H2 O2 production. These results show that the apparent monooxygenase activity observed in SscLPMO10B reactions without exogenously added H2 O2 reflects a peroxygenase reaction.

1H, 13C, 15N resonance assignment of the apo form of the small, chitin-active lytic polysaccharide monooxygenase JdLPMO10A from Jonesia denitrificans.

The lytic polysaccharide monooxygenase JdLPMO10A is the N-terminal domain of the multimodular protein Jd1381. The isolated JdLPMO10A domain is one of the smallest chitin-active lytic polysaccharide monooxygenases known to date with a size of only 15.5 kDa. JdLPMO10A is a copper-dependent oxidative enzyme that depolymerizes chitin by hydroxylating the C1 carbon in the glycosidic bond. JdLPMO10A has been isotopically labeled and recombinantly expressed. Here, we report the 1H, 13C, 15N resonance assignment of JdLPMO10A. Secondary structural elements predicted based on the NMR assignment are in excellent agreement with the crystal structure of JdLPMO10A.