Fungal lytic polysaccharide monooxygenases bind starch and β‐cyclodextrin similarly to amylolytic hydrolases

Abstract

Edited by Judit Ov adiStarch-binding modules of family 20 (CBM20) are present in 60% of lytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative breakdown of starch, which highlights functional importance in LPMO activity. The substrate-binding properties of starch-active LMPOs, however, are currently unexplored. Affinities and binding-thermodynamics of two recombinant fungal LPMOs toward starch and b-cyclodextrin were shown to be similar to fungal CBM20s. Amplex Red assays showed ascorbate and Cu-dependent activity, which was inhibited in the presence of b-cylodextrin and amylose. Phylogenetically, the clustering of CBM20s from starch-targeting LPMOs and hydrolases was in accord with taxonomy and did not correlate to appended catalytic activity. Altogether, these results demonstrate that the CBM20-binding scaffold is retained in the evolution of hydrolytic and oxidative starch-degrading activities. Keywords: AA13;carbohydrate-binding module; CBM20; lytic polysaccharide monooxygenase; starch binding; b-cyclodextrin Starch is a major renewable energy storage polysaccharide in plants and an important resource not only as a food but also as an industrial feedstock in biofuels, pharmaceuticals, detergents, and cosmetics [1][2][3]. Starch consists of two types of homo-glucose polymers: the mainly linear a-1,4-linked amylose and amylopectin, constituting 65-82% (w/w) of the starch granule and differing from amylose by having a larger molecular mass and roughly 5% a-1,6-branches of 12-15 glucosyl units long on average [2,4,5]. Starch is biosynthesized as insoluble granules, varying in size, morphology, crystal packing, and crystallinity that ranges from 15 to 45% depending on botanical origin [6][7][8]. Radially alternating amorphous and semicrystalline layers in the starch granule arise from the packing of double helices formed by adjacent branches in amylopectin, with the semicrystalline regions contributing to resistance of starch to enzymatic degradation [9,10]. Many industrial applications require the disruption of starch granules through hydrothermal, harsh chemical, or enzymatic treatments [11][12][13]. Despite development of relatively efficient a-amylases and other starch-degrading enzymes, there is still a significant margin for improving starch hydrolysis yields and shortening processing time, which would significantly reduce energy and costs of the process [14,15].Typically, glycoside hydrolases (GHs) that degrade complex polysaccharides possess carbohydrate-binding modules (CBMs) that promote enzyme-substrate proximity and thereby enhance catalytic efficiency [16,17]. Moreover, CBMs can also modulate the specificity and activity of cognate enzymes against plant cell wall Abbreviations AA, auxiliary activity; CAZy, carbohydrate-active enzymes; CBM, carbohydrate-binding module; DSC, differential scanning calorimetry; GH, glycoside hydrolase; ITC, isothermal titration calorimetry; LPMO, lytic polysaccharide monooxygenase; SBS, starch-binding site; SDS/PAGE, sodium dodecyl sulfate-polyacrylamide ge...

The starch‐binding domain family CBM41—An in silico analysis of evolutionary relationships

“…Overall, it can be concluded that as originally observed for SBDs from the family CBM20 from various amylolytic GH families and recently confirmed also for this SBD present in non‐amylolytic starch‐degrading lytic polysaccharide monooxygenases, taxonomy seems to be more respected in the evolution of CBMs than the specificity of the enzyme to which a given CBM sequence is attached.…”

Substrate selectivity in starch polysaccharide monooxygenases

“…Because different structural analysis tools give different results in H-bond determination, a range in the number of H-bonds formed between the AA13 PMO and each substrate is reported, representing the H-bonds identified using a variety of analyses. A random amylose coil forms 16 -19 H-bonds with 11 residues of AA13 PMOs (Cys 25 , Ile 27 , Glu 29 , Arg 44 , Ala 52 , Arg 53 , Asn 88 , Asp 90 , Pro 172 , Lys 173 , and Gln 219 ) (Fig. S6).…”

Interaction of carbohydrate binding module 20 with starch substrates

“…These values are similar to those obtained experimentally for CBM20 complexes with b-cyclodextrin and some oligosaccharide. 18,45 For all three substrates, the binding affinity at BdS1 is signicantly larger (by 1.8-3.5 kcal mol À1 ) than that at BdS2. The average binding affinity of both binding sites deduced for ADH10, ASH10, and ASC10 is À10.9 AE 0.5, À10.2 AE 0.3, and À5.6 AE 0.3 kcal mol À1 .…”

“…4,6 Several isothermal calorimetry studies revealed 5-8 kcal mol À1 binding affinities for oligosaccharides of the CBM20s of the glucoamylase from Aspergillus niger 45 and starchactive polysaccharide monooxygenases (AA13) from Magnaporthe oryzae and Aspergillus terreus. 18 Moderate affinity for starch granules were also demonstrated for these CBM20s. Given the moderate affinity of CBM20 for starch, we have been able to use CBM20 as an affinity tag for facile purication of NCU08746, an AA13 polysaccharide monooxygenase from Neurospora crassa (NcAA13), using an amylose resin column.…”

“…During this period, the interests in the role of CBMs in substrate binding and catalysis of carbohydrate-active enzymes were also reignited. [17][18][19][20][21] CBM20 was found as a C-terminal domain in the majority of starchactive PMOs (AA13), [22][23][24] the only PMO family that oxidatively cleaves on a-glycosidic bond in starch. CBM20 appears to have signicant roles in the activity of AA13 PMOs on various type of starch.…”