Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

Abstract

The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

Regulation of Gene Expression during the Onset of Ligninolytic Oxidation by Phanerochaete chrysosporium on Spruce Wood

“…One of 16 P. chrysosporium genes encoding a lytic polysaccharide monooxygenase (LPMO) was upregulated more than 4-fold at 96 h. Formerly classified as glycoside hydrolases (50), these enzymes are now known for their oxidative attack on cellulose and xylans (51)(52)(53)(54)(55). The current protein model for the upregulated LPMO (Phach ID 2934397) lacks a secretion signal, but the alternative model given for it in the v2.1 database (Phach ID122129) features a complete N terminus with a predicted secretion signal.…”

Spectroscopic and computational insight into the activation of O 2 by the mononuclear Cu center in polysaccharide monooxygenases

“…It is interesting to note that in a recently published study by Isaksen et al (38), hydrogen peroxide formation, by a Cu-AA9 enzyme with different reductants and O 2 , was detected only in the absence of substrate or with substrates that were not subject to AA9 activity. If the superoxide is indeed stabilized by the substrate, it may be able to directly attack the polysaccharide, or it may be further reduced to a more reactive species by either small molecules or cellobiose dehydrogenase (a known AA9 reducing cofactor) (7,13,39). This awaits further experimental investigation.…”

Plant-Polysaccharide-Degrading Enzymes from Basidiomycetes

“…Currently, cellulose-cleaving activities of LPMOs have been biochemically confirmed only for GH61D of the white rot fungus P. chrysosporium (PcGH61D) (125); the above-mentioned ascomycete LPMOs from H. jecorina, T. terrestris, T. aurantiacus, and N. crassa; and GH61A and GH61B of the ascomycete fungus P. anserina (27,28,31,32,126,127). PcGH61D is not active on soluble cellooligosaccharides (125), but it is able to oxidize phosphoric acid-swollen cellulose in the presence of ascorbic acid and to release lactone, which is spontaneously converted to aldonic acid (125).…”