Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme’s active site yields insights into the mechanism of action of this important class of enzymes.

WITHDRAWN: Identification of an endogenous redox partner for lytic polysaccharide monooxygenase-based oxidative cleavage of polysaccharides

“…Therefore, in order to find out substrate preference of BcLPMO 9 C, we investigated cleavage of different cellooligosaccharides, ranging from cello -triose, -tetraose, -pentaose and -hexaose, by rBcLPMO 9 C in presence of ascorbate as an electron donor. rBcLPMO 9 C was able to oxidatively cleave cellohexaose preferentially yielding cellotriose, cellobiose and cellobionic acid (Fig 3C), whereas BcLPMO 9 C was inactive on shorter cello-oligosaccharides, such as cellotriose, -tetraose, -pentaose (data not shown), suggesting that the enzyme requires at least six glucosyl units for productive substrate binding and degradation. In order to assess enzymatic activity of rBcLPMO 9 C quantitatively, we estimated specific activity of recombinant rBcLPMO 9 C using amplex red assay as described earlier (29).…”

“…Thus, in pursuit of identifying the interacting electron-donor partner for LPMO, we used LC-MS based pull-down analysis. Pull down assay using GST-tagged BcLPMO 9 (Table 1). In a parallel set of experiment, we carried out label-transfer assay as defined previously (47), wherein we used trifunctional cross linker-tagged rBcLPMO 9 C and supernatant concentrate from B. cinerea.…”

“…Pull down assay using GST-tagged BcLPMO 9 (Table 1). In a parallel set of experiment, we carried out label-transfer assay as defined previously (47), wherein we used trifunctional cross linker-tagged rBcLPMO 9 C and supernatant concentrate from B. cinerea. The transfer of biotin tag from rBcLPMO 9 C to interacting protein was detected using streptavidin conjugated to AP (Fig 4A).…”

“…under Carbohydrate Active Enzymes (CAZy) database as auxillary activity families (AA9-AA11 and AA13) wherein AA9 catalyze scission of glycosidic bonds in cellulose chain (7) and AA10-11 are responsible for oxidative degradation of mainly chitins whereas AA13 plays important role in starch degradation (7)(8)(9)(10)(11). LPMOs can catalyze cleavage at C-1/4 position and thus are divided into three categories depending on their regioselectivity: Type I mediates cleavage at C-1 position; Type II does oxidative degradation at C-4 position whereas Type III enzymes creates scission at both C-1 and C-4 positions (11).…”

Backbone and side-chain 1H, 13C, and 15N chemical shift assignments for the apo-form of the lytic polysaccharide monooxygenase NcLPMO9C

“…Enzymatic biomass conversion normally requires several enzymes, including hydrolases and the recently discovered lytic polysaccharide monooxygenases (LPMOs). LPMOs comprise four families of carbohydrate-active enzymes (AA9, AA10, AA11 and AA13) (Levasseur et al 2013;Hemsworth et al 2014;Lo Leggio et al 2015;Beeson et al 2015;Hemsworth et al 2015) that catalyze oxidative cleavage (Vaaje-Kolstad et al 2010;Quinlan et al 2011;Phillips et al 2011;Kim et al 2014). LPMOs boost the activity of the hydrolytic polysaccharide degrading enzymes and are thus of great importance for efficient biomass conversion.…”