Substrate selectivity in starch polysaccharide monooxygenases

Vu, Van V.; Detomasi, Tyler C.; Henry, Skylar J.W.; Ngo, Son Tung; Span, Elise A.; Marletta, Michael A.

doi:10.1074/jbc.ra119.009509

Cited by 39 publications

(43 citation statements)

References 67 publications

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“…This affinity for β-cylcodextrin (and starch) is not provided by the active site, as the An AA13B homologue lacking the CBM20 was not captured by the β-cyclodextrin affinity column [ 13 ]. The active site of AA13 LPMOs has been proposed to recognize the helical conformation of starch in a shallow groove [ 7 , 28 ], albeit likely with a much lower affinity than the CBM20-mediated binding as argued above. A possible advantage of the An AA13B is that the activity is less localized due to the more dynamic lower affinity binding and smaller size as compared to An AA13A, which may allow deeper penetration in the starch granule.…”

Section: Discussionmentioning

confidence: 99%

Loss of AA13 LPMOs impairs degradation of resistant starch and reduces the growth of Aspergillus nidulans

Momeni

Leth

Sternberg

et al. 2020

Biotechnol Biofuels

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Background Lytic polysaccharide monooxygenases (LPMOs) are often studied in simple models involving activity measurements of a single LPMO or a blend thereof with hydrolytic enzymes towards an insoluble substrate. However, the contribution of LPMOs to polysaccharide breakdown in complex cocktails of hydrolytic and oxidative enzymes, similar to fungal secretomes, remains elusive. Typically, two starch-specific AA13 LPMOs are encoded by mainly Ascomycota genomes. Here, we investigate the impact of LPMO loss on the growth and degradation of starches of varying resistance to amylolytic hydrolases by Aspergillus nidulans . Results Deletion of the genes encoding An AA13A that possesses a CBM20 starch-binding module, An AA13B (lacking a CBM20) or both AA13 genes resulted in reduced growth on solid media with resistant, but not soluble processed potato starch. Larger size and amount of residual starch granules were observed for the AA13-deficient strains as compared to the reference and the impairment of starch degradation was more severe for the strain lacking An AA13A based on a microscopic analysis. After 5 days of growth on raw potato starch in liquid media, the mount of residual starch was about fivefold higher for the AA13 gene deletion strains compared to the reference, which underscores the importance of LPMOs for degradation of especially resistant starches. Proteomic analyses revealed substantial changes in the secretomes of the double AA13 gene deletion, followed by the An AA13A-deficient strain, whereas only a single protein was significantly different in the proteome of the An AA13B-deficient strain as compared to the reference. Conclusions This study shows that the loss of AA13, especially the starch-binding An AA13A, impairs degradation of resistant potato starch, but has limited impact on less-resistant wheat starch and no impact on processed solubilized starch. The effects of LPMO loss are more pronounced at the later stages of fungal growth, likely due to the accumulation of the less-accessible regions of the substrate. The striking impairment in granular starch degradation due to the loss of a single LPMO from the secretome offers insight into the crucial role played by AA13 in the breakdown of resistant starch and presents a methodological framework to analyse the contribution of distinct LPMOs towards semi-crystalline polysaccharides under in vivo conditions.

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Section: Discussionmentioning

confidence: 99%

Loss of AA13 LPMOs impairs degradation of resistant starch and reduces the growth of Aspergillus nidulans

Momeni

Leth

Sternberg

et al. 2020

Biotechnol Biofuels

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“…It may thus seem that nature has evolved different strategies for LPMOs to have affinity for their substrates, but this is not yet sufficiently supported by systematic experimental studies. Existing data show that the CBMs of LPMOs have the same function as in GHs [53,66,72,110,111,192,209,371] and it has also been shown that, like for GHs, the presence of a CBM becomes less important, and even unfavorable, when running reactions at high substrate concentrations [66].…”

Section: The Role Of Cbms: For Cellulases Hemicellulases and Lpmosmentioning

confidence: 99%

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

Østby

Hansen

Horn

et al. 2020

Journal of Industrial Microbiology and Biotechnology

129

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Efficient saccharification of lignocellulosic biomass requires concerted development of a pretreatment method, an enzyme cocktail and an enzymatic process, all of which are adapted to the feedstock. Recent years have shown great progress in most aspects of the overall process. In particular, increased insights into the contributions of a wide variety of cellulolytic and hemicellulolytic enzymes have improved the enzymatic processing step and brought down costs. Here, we review major pretreatment technologies and different enzyme process setups and present an in-depth discussion of the various enzyme types that are currently in use. We pay ample attention to the role of the recently discovered lytic polysaccharide monooxygenases (LPMOs), which have led to renewed interest in the role of redox enzyme systems in lignocellulose processing. Better understanding of the interplay between the various enzyme types, as they may occur in a commercial enzyme cocktail, is likely key to further process improvements.

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“…Since Phe166 cannot flip out away from the active site in the crystal, alternative motions are observed in the crystal in response to loss of metal binding. Interestingly, Phe166 marks the beginning of the 'variable substrate interaction region' in the most recent article on AA13 [40], which almost perfectly coincides with the most flexible part of the structure in both simulations (residues 166-170) and thus these dynamics are likely to be of functional importance. A recent study of dynamics of LPMOs [43] by the computationally cheaper Elastic Network Model method, identified two different regions of correlated dynamics in AoAA13 (residues 206-213 and 213-221).…”

Section: Resultsmentioning

confidence: 70%

“…Activity has not been demonstrated for this particular LPMO, but the presumed binding surface near the active site is highly conserved in this family (Fig S1; sequences analyzed with the ConSurf server [39]). In the last stages of completion of this manuscript, a detailed study of substrate preference of two AA13 LPMOs, one naturally with and one without CBM, has been published [40]. Based on the pattern of degradation and computational docking using the structure of AoAA13 (the only one still available for this family), the authors suggest that maltooctaose may be the minimal substrate and furthermore suggest that the preferred substrate is an amylose double helix.…”

Section: Resultsmentioning

confidence: 99%

Further structural studies of the lytic polysaccharide monooxygenase AoAA13 belonging to the starch-active AA13 family

Muderspach

Tandrup

Frandsen

et al. 2019

Amylase

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Lytic polysaccharide monooxygenases (LPMOs) are recently discovered copper enzymes that cleave recalcitrant polysaccharides by oxidation. The structure of an Aspergillus oryzae LPMO from the starch degrading family AA13 (AoAA13) has previously been determined from an orthorhombic crystal grown in the presence of copper, which is photoreduced in the structure. Here we describe how crystals reliably grown in presence of Zn can be Cu-loaded post crystallization. A partly photoreduced structure was obtained by severely limiting the X-ray dose, showing that this LPMO is much more prone to photoreduction than others. A serial synchrotron crystallography structure was also obtained, showing that this technique may be promising for further studies, to reduce even further photoreduction. We additionally present a triclinic structure of AoAA13, which has less occluded ligand binding site than the orthorhombic one. The availability of the triclinic crystals prompted new ligand binding studies, which lead us to the conclusion that small starch analogues do not bind to AoAA13 to an appreciable extent. A number of disordered conformations of the metal binding histidine brace have been encountered in this and other studies, and we have previously hypothesized that this disorder may be a consequence of loss of copper. We performed molecular dynamics in the absence of active site metal, and showed that the dynamics in solution differ somewhat from the disorder observed in the crystal, though the extent is equally dramatic.

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Substrate selectivity in starch polysaccharide monooxygenases

Cited by 39 publications

References 67 publications

Loss of AA13 LPMOs impairs degradation of resistant starch and reduces the growth of Aspergillus nidulans

Loss of AA13 LPMOs impairs degradation of resistant starch and reduces the growth of Aspergillus nidulans

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

Further structural studies of the lytic polysaccharide monooxygenase AoAA13 belonging to the starch-active AA13 family

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