ABSTRACT: Background:Polysaccharide monooxygenases (PMOs) of the auxiliary activity 9 (AA9) family have been reported to oxidize C1, C4, and C6 positions in cellulose. However, currently no direct evidence exists that PMOs oxidize C6 positions in cellulose, and molecular mechanism of C1, C4 and C6 oxidation is unclear. Results:In this study, a PMO gene (Ctpmo1) belonging to AA9 was isolated from Chaetomium thermophilum and successfully expressed and correctly processed in Pichia pastoris. A simple and effective chemical method of using Br2 to oxidize CtPMO1 reaction products was developed to directly identify C4- and C6-oxidized products by matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF-MS). The PMO (CtPMO1) cleaves phosphoric acid-swollen cellulose (PASC) and celloheptaose, resulting in the formation of oxidized and nonoxidized oligosaccharides. Product identification shows that the enzyme can oxidize C1, C4, and C6 in PASC and cello-oligosaccharides. Mutagenesis of the aromatic residues Tyr27, His64, His157 and residue Tyr206 on the flat surface of CtPMO1 was carried out using site-directed mutagenesis to form the mutated enzymes Y27A, H64A, H157A, and Y206A. It was demonstrated that Y27A retained complete activity of C1, C4, and C6 oxidation on cellulose; Y206A retained partial activity of C1 and C4 oxidation but completely lost activity of C6 oxidation on cellulose; H64A almost completely lost activity of C1, C4, and C6 oxidation on cellulose; and H157A completely lost activity of C1, C4, and C6 oxidation on cellulose. Conclusions:This finding provides direct and molecular evidence for C1, C4, especially C6 oxidation by lytic polysaccharide monooxygenase. CtPMO1 oxidizes not only C1 and C4 but also C6 positions in cellulose. The aromatic acid residues His64, His157 and residue Tyr206 on CtPMO1 flat surface are involved in activity of C1, C4, C6 oxidation.
