A family of starch-active polysaccharide monooxygenases

Abstract

The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries.copper enzymes | oxygen activation | CBM20 P olysaccharide monooxygenases (PMOs) are enzymes secreted by a variety of fungal and bacterial species (1-5). They have recently been found to oxidatively degrade chitin (6-8) and cellulose (8)(9)(10)(11)(12)(13)(14). PMOs have been shown to oxidize either the C1 or C4 atom of the β(1→4) glycosidic bond on the surface of chitin (6, 7) or cellulose (10)(11)(12)14), resulting in the cleavage of this bond and the creation of new chain ends that can be subsequently processed by hydrolytic chitinases and cellulases. Several fungal PMOs were shown to significantly enhance the degradation of cellulose by hydrolytic cellulases (9), indicating that these enzymes can be used in the conversion of plant biomass into biofuels and other renewable chemicals.There are three families of PMOs characterized thus far: fungal PMOs that oxidize cellulose (9-12) (also known as GH61 and AA9); bacterial PMOs that are active either on chitin (6, 8) or cellulose (8, 13) (also known as CBM33 and AA10); and fungal PMOs that oxidize chitin (AA11) (7). Sequence homology between these three families is very low. Nevertheless, the available structures of PMOs from all three families reveal a conserved fold, including an antiparallel β-sandwich core and a highly conserved ...

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

“…Fungal LPMOs that oxidize cellulose belong to AA9 (formerly GH61) (315,319,321), and more recently a fungal LPMO that oxidizes chitin has been characterized for AA11 (322). In addition, an LPMO active on starch was reported and is now classified as AA13 (323). Chitin and cellulose are ␤(1¡4)-linked polymers with flat molecular surfaces, whereas starch contains ␣(1¡4) linkages (amylose) and ␣(1¡6)-linked side chains (amylopectin).…”

“…LPMOs have been shown to oxidize either the C-1 or C-4 atom of the ␤(1¡4) glycosidic bond on the surface of chitin or cellulose (315,317,318) and C-1 of starch (323), resulting in the cleavage of this bond and the creation of new chain ends that can be subsequently processed by hydrolytic chitinases and cellulases. Already before the mode of action of LPMOs was recognized, it was shown that the addition of these proteins (i.e., GH61 and CBM33 proteins) to enzyme mixtures significantly enhanced the degradation of cellulose and chitin in synergism with the respective hydrolases (324)(325)(326).…”

Activity, stability and 3-D structure of the Cu(ii) form of a chitin-active lytic polysaccharide monooxygenase from Bacillus amyloliquefaciens

“…These enzymes are the lytic polysaccharide monooxygenases (LPMOs), which are currently sequenceclassified into four families in the CAZy database (www.CAZy.org) -AA9 and AA10 (previously GH61 and CBM33 respectively) 3 , as well as the more recent AA11 4 and AA13 5,6 .…”

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

“…However, it was noted that the abundance, the large sequence variation, and the varying domain composition of LPMOs encoded in the genomes of biomass-degrading microorganisms suggest that some of these enzymes could have other substrates (1). Indeed, three studies published in 2014 have broadened the LPMO paradigm by demonstrating cleavage of soluble cellodextrins (13), hemicelluloses with ␤(134)-glucan backbones (14), and even starch (15). Based on sequence characteristics, LPMOs are currently categorized in auxiliary activity (AA) families 9 -11 and 13 of the CAZy database (16).…”