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Substrate selectivity in starch polysaccharide monooxygenases.

ABSTRACT: Degradation of polysaccharides is central to numerous biological and industrial processes. Starch-active polysaccharide monooxygenases (AA13 PMOs) oxidatively degrade starch and can potentially be used with industrial amylases to convert starch into a fermentable carbohydrate. The oxidative activities of the starch-active PMOs from the fungi Neurospora crassa and Myceliophthora thermophila, NcAA13 and MtAA13, respectively, on three different starch substrates are reported here. Using high-performance anion-exchange chromatography coupled with pulsed amperometry detection, we observed that both enzymes have significantly higher oxidative activity on amylose than on amylopectin and cornstarch. Analysis of the product distribution revealed that NcAA13 and MtAA13 more frequently oxidize glycosidic linkages separated by multiples of a helical turn consisting of six glucose units on the same amylose helix. Docking studies identified important residues that are involved in amylose binding and suggest that the shallow groove that spans the active-site surface of AA13 PMOs favors the binding of helical amylose substrates over nonhelical substrates. Truncations of NcAA13 that removed its native carbohydrate-binding module resulted in diminished binding to amylose, but truncated NcAA13 still favored amylose oxidation over other starch substrates. These findings establish that AA13 PMOs preferentially bind and oxidize the helical starch substrate amylose. Moreover, the product distributions of these two enzymes suggest a unique interaction with starch substrates.

SUBMITTER: Vu VV

PROVIDER: S-EPMC6690692 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Substrate selectivity in starch polysaccharide monooxygenases.

Vu Van V VV Hangasky John A JA Detomasi Tyler C TC Henry Skylar J W SJW Ngo Son Tung ST Span Elise A EA Marletta Michael A MA

The Journal of biological chemistry 20190624 32

Degradation of polysaccharides is central to numerous biological and industrial processes. Starch-active polysaccharide monooxygenases (AA13 PMOs) oxidatively degrade starch and can potentially be used with industrial amylases to convert starch into a fermentable carbohydrate. The oxidative activities of the starch-active PMOs from the fungi Neurospora crassa and Myceliophthora thermophila, NcAA13 and MtAA13, respectively, on three different starch substrates are repo ...[more]

PMID: 31235519

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Project description:BackgroundUnderstanding the diversity of lignocellulose-degrading enzymes in nature will provide insights for the improvement of cellulolytic enzyme cocktails used in the biofuels industry. Two families of enzymes, fungal AA9 and bacterial AA10, have recently been characterized as crystalline cellulose or chitin-cleaving lytic polysaccharide monooxygenases (LPMOs). Here we analyze the sequences, structures, and evolution of LPMOs to understand the factors that may influence substrate specificity both within and between these enzyme families.ResultsComparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families demonstrated that, although these two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues. Phylogenetic analysis of the AA10 family identified clades with putative chitinolytic and cellulolytic activities. Estimation of the rate of synonymous versus non-synonymous substitutions (dN/dS) within two major AA10 subclades showed distinct selective pressures between putative cellulolytic genes (subclade A) and CBP21-like chitinolytic genes (subclade D). Estimation of site-specific selection demonstrated that changes in the active sites were strongly negatively selected in all subclades. Furthermore, all codons in the subclade D had dN/dS values of less than 0.7, whereas codons in the cellulolytic subclade had dN/dS values of greater than 1.5. Positively selected codons were enriched at sites localized on the surface of the protein adjacent to the active site.ConclusionsThe structural similarity but absence of significant sequence similarity between AA9 and AA10 families suggests that these enzyme families share an ancient ancestral protein. Combined analysis of amino acid sites under Darwinian selection and structural homology modeling identified a subclade of AA10 with diversifying selection at different surfaces, potentially used for cellulose-binding and protein-protein interactions. Together, these data indicate that AA10 LPMOs are under selection to change their function, which may optimize cellulolytic activity. This work provides a phylogenetic basis for identifying and classifying additional cellulolytic or chitinolytic LPMOs.

Project description:BackgroundLytic polysaccharide monooxgygenases (LPMOs) are known to boost the hydrolytic breakdown of lignocellulosic biomass, especially cellulose, due to their oxidative mechanism. For their activity, LPMOs require an electron donor for reducing the divalent copper cofactor. LPMO activities are mainly investigated with ascorbic acid as a reducing agent, but little is known about the effect of plant-derived reducing agents on LPMOs activity.ResultsHere, we show that three LPMOs from the fungus Myceliophthora thermophila C1, MtLPMO9A, MtLPMO9B and MtLPMO9C, differ in their substrate preference, C1-/C4-regioselectivity and reducing agent specificity. MtLPMO9A generated C1- and C4-oxidized, MtLPMO9B C1-oxidized and MtLPMO9C C4-oxidized gluco-oligosaccharides from cellulose. The recently published MtLPMO9A oxidized, next to cellulose, xylan, β-(1 → 3, 1 → 4)-glucan and xyloglucan. In addition, MtLPMO9C oxidized, to a minor extent, xyloglucan and β-(1 → 3, 1 → 4)-glucan from oat spelt at the C4 position. In total, 34 reducing agents, mainly plant-derived flavonoids and lignin-building blocks, were studied for their ability to promote LPMO activity. Reducing agents with a 1,2-benzenediol or 1,2,3-benzenetriol moiety gave the highest release of oxidized and non-oxidized gluco-oligosaccharides from cellulose for all three MtLPMOs. Low activities toward cellulose were observed in the presence of monophenols and sulfur-containing compounds.ConclusionsSeveral of the most powerful LPMO reducing agents of this study serve as lignin building blocks or protective flavonoids in plant biomass. Our findings support the hypothesis that LPMOs do not only vary in their C1-/C4-regioselectivity and substrate specificity, but also in their reducing agent specificity. This work strongly supports the idea that the activity of LPMOs toward lignocellulosic biomass does not only depend on the ability to degrade plant polysaccharides like cellulose, but also on their specificity toward plant-derived reducing agents in situ.

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Substrate selectivity in starch polysaccharide monooxygenases.

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Substrate selectivity in starch polysaccharide monooxygenases.

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