Sebastian J. Muderspach

University of Copenhagen

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Leila Lo Leggio

University of Copenhagen

Jens-Christian N. Poulsen

University of Copenhagen

Kristian B. R. M. Krogh

Novozymes (Denmark)

Tobias Tandrup

Technical University of Denmark

Kristian E. H. Frandsen

University of Copenhagen

Sanchari Banerjee

SUNY Upstate Medical University

Morten H. H. Nørholm

Technical University of Denmark

JØ

Johan Ø. Ipsen

University of Copenhagen

Kenneth Jensen

Novozymes (Denmark)

Cristina Hernández‐Rollán

Technical University of Denmark

Cooperative Institutions

University of Copenhagen

Technical University of Denmark

Institute of Chemistry of the Slovak Academy of Sciences

Slovak Academy of Sciences

Novo Nordisk Foundation

Novozymes (Denmark)

Universidade de São Paulo

Wageningen University & Research

Universidade Federal de São Carlos

Aarhus University

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Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding

IUCrJ (2022)

Tobias Tandrup Sebastian J. Muderspach Sanchari Banerjee Gianluca Santoni Johan Ø. Ipsen

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes capable of degrading polysaccharide substrates oxidatively. The generally accepted first step in the LPMO reaction is the reduction of the active-site metal ion from Cu 2+ to Cu + . Here we have used a systematic diffraction data collection method to monitor structural changes in two AA9 LPMOs, one from Lentinus similis ( Ls AA9_A) and one from Thermoascus aurantiacus ( Ta AA9_A), as the active-site Cu is photoreduced in the X-ray beam. For Ls AA9_A, the protein produced in two different recombinant systems was crystallized to probe the effect of post-translational modifications and different crystallization conditions on the active site and metal photoreduction. We can recommend that crystallographic studies of AA9 LPMOs wishing to address the Cu 2+ form use a total X-ray dose below 3 × 10 4 Gy, while the Cu + form can be attained using 1 × 10 6 Gy. In all cases, we observe the transition from a hexacoordinated Cu site with two solvent-facing ligands to a T-shaped geometry with no exogenous ligands, and a clear increase of the θ 2 parameter and a decrease of the θ 3 parameter by averages of 9.2° and 8.4°, respectively, but also a slight increase in θ T . Thus, the θ 2 and θ 3 parameters are helpful diagnostics for the oxidation state of the metal in a His-brace protein. On binding of cello-oligosaccharides to Ls AA9_A, regardless of the production source, the θ T parameter increases, making the Cu site less planar, while the active-site Tyr—Cu distance decreases reproducibly for the Cu 2+ form. Thus, the θ T increase found on copper reduction may bring Ls AA9_A closer to an oligosaccharide-bound state and contribute to the observed higher affinity of reduced Ls AA9_A for cellulosic substrates.

Lytic cycle

10.1107/s2052252522007175

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Citations (17)

Structure ofAspergillus aculeatusβ-1,4-galactanase in complex with galactobiose

Acta Crystallographica Section F Structural Biology Communications (2019)

Søs Torpenholt Jens-Christian N. Poulsen Sebastian J. Muderspach Leonardo De Maria Leila Lo Leggio

β-1,4-Galactanases are glycoside hydrolases that are involved in the degradation of pectin and belong to family 53 in the classification of glycoside hydrolases. Previous studies have elucidated the structures of several fungal and two bacterial galactanases, while biochemical studies have indicated differences in the product profiles of different members of the family. Structural studies of ligand complexes have to date been limited to the bacterial members of the family. Here, the first structure of a fungal galactanase in complex with a disaccharide is presented. Galactobiose binds to subsites −1 and −2, thus improving our understanding of ligand binding to galactanases.

10.1107/s2053230x19005612

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Citations (6)

ASPERGILLUS ACULEATUS GALACTANASE

Sebastian J. Muderspach Søs Torpenholt Leila Lo Leggio Jens-Christian N. Poulsen

10.2210/pdb6q3r/pdb

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Citations (1)

Protonation State of an Important Histidine from High Resolution Structures of Lytic Polysaccharide Monooxygenases

Biomolecules (2022)

Sanchari Banerjee Sebastian J. Muderspach Tobias Tandrup Kristian E. H. Frandsen Raushan Kumar Singh

Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the Nε2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

Glycosidic bond

10.3390/biom12020194

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Citations (25)

Contributors

Elsevier eBooks (2023)

Shadab Ahmed Rudrarup Bhattacharjee Arindam Bhattacharyya Peter Biely Caio Cesar de Mello Capetti

10.1016/b978-0-323-91805-3.09989-5

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Citations (0)

Structure, function, and protein engineering of GH53 β-1,4-galactanases

Elsevier eBooks (2023)

Sebastian J. Muderspach Kenneth Jensen Kristian B. R. M. Krogh Leila Lo Leggio

10.1016/b978-0-323-91805-3.00018-6

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Citations (1)

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

Amylase (2019)

Sebastian J. Muderspach Tobias Tandrup Kristian E. H. Frandsen Gianluca Santoni Jens-Christian N. Poulsen

Abstract 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 ( Ao AA13) 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 Ao AA13, 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 Ao AA13 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.

Triclinic crystal system

Orthorhombic crystal system

Lytic cycle

10.1515/amylase-2019-0004

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Citations (4)

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

Amylase (2019)

Sebastian J. Muderspach Tobias Tandrup Kristian E. H. Frandsen Gianluca Santoni Jens-Christian N. Poulsen

Lytic cycle

10.2210/pdb6tbr/pdb

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Engineering the substrate binding site of the hyperthermostable archaeal endo-β-1,4-galactanase from Ignisphaera aggregans

Biotechnology for Biofuels (2021)

Sebastian J. Muderspach Folmer Fredslund Verena Volf Jens-Christian N. Poulsen Thomas Blicher

Abstract Background Endo-β-1,4-galactanases are glycoside hydrolases (GH) from the GH53 family belonging to the largest clan of GHs, clan GH-A. GHs are ubiquitous and involved in a myriad of biological functions as well as being widely used industrially. Endo-β-1,4-galactanases, in particular hydrolyse galactan and arabinogalactan in pectin, a major component of the primary plant cell wall, with important functions in plant defence and application in the food and other industries. Here, we explore the family’s biological diversity by characterizing the first archaeal and hyperthermophilic GH53 galactanase, and utilize it as a scaffold for engineering enzymes with different product lengths. Results A galactanase gene was identified in the genome of the anaerobic hyperthermophilic archaeon Ignisphaera aggregans , and the isolated catalytic domain expressed and characterized (IaGal). IaGal presents the typical (βα) 8 barrel structure of clan GH-A enzymes, with catalytic carboxylates at the end of the 4th and 7th barrel strands. Its activity optimum of at least 95 °C and melting point over 100 °C indicate extreme thermostability, a very advantageous property for industrial applications. If enzyme depletion is reduced, so is the need for re-addition, and thus costs. The main stabilizing features of IaGal compared to other structurally characterized members are π–π and cation–π interactions. The length of the substrate binding site—and thus produced oligosaccharide products—is intermediate compared to previously characterized galactanases. Variants inspired by the structural diversity in the GH53 family were rationally designed to shorten or extend the substrate binding groove, in order to modulate product length. Subsite-deleted variants produced shorter products than IaGal, as do the fungal galactanases inspiring the design. IaGal variants engineered with a longer binding site produced a less expected degradation pattern, though still different from that of wild-type IaGal. All variants remained extremely stable. Conclusions We have characterized in detail the most thermophilic endo-β-1,4-galactanase known to date and successfully engineered it to modify the degradation profile, while maintaining much of its desirable thermostability. This is an important achievement as oligosaccharide products length is an important property for industrial and natural GHs alike.

Thermostability

Protein Engineering

Arabinogalactan

Cellulosome

10.1186/s13068-021-02025-6

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Citations (7)

Copper binding and reactivity at the histidine brace motif: insights from mutational analysis of the Pseudomonas fluorescens copper chaperone CopC

FEBS Letters (2021)

Johan Ø. Ipsen Cristina Hernández‐Rollán Sebastian J. Muderspach Søren Brander Andreas Birk Bertelsen

The histidine brace (His‐brace) is a copper‐binding motif that is associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical and structural methods to characterize mutants of a His‐brace‐containing copper chaperone from Pseudomonas fluorescens (PfCopC). A total of 15 amino acid variants in primary and second‐sphere residues were produced and characterized in terms of their copper binding and redox properties. PfCopC has a very high affinity for Cu(II) and also binds Cu(I). A high reorganization barrier likely prevents redox cycling and, thus, catalysis. In contrast, mutations in the conserved second‐sphere Glu27 enable slow oxidation of ascorbate. The crystal structure of the variant E27A confirmed copper binding at the His‐brace. Unexpectedly, Asp83 at the equatorial position was shown to be indispensable for Cu(II) binding in the His‐brace of PfCopC. A PfCopC mutant that was designed to mimic the His‐brace from lytic polysaccharide monooxygenase‐like family X325 did not bind Cu(II), but was still able to bind Cu(I). These results highlight the importance of the proteinaceous environment around the copper His‐brace for reactivity and, thus, the difference between enzyme and chaperone.

Pseudomonas fluorescens

Chaperone (clinical)

10.1002/1873-3468.14092

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Citations (7)