Andaleeb H et al. (2020), High-resolution crystal structure and biochemic...

ECB-ART-49586

Sci Rep 2020 Sep 24;101:15658. doi: 10.1038/s41598-020-72644-w.

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High-resolution crystal structure and biochemical characterization of a GH11 endoxylanase from Nectria haematococca.

Andaleeb H , Ullah N , Falke S , Perbandt M , Brognaro H , Betzel C .

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Enzymatic degradation of vegetal biomass offers versatile procedures to improve the production of alternative fuels and other biomass-based products. Here we present the three-dimensional structure of a xylanase from Nectria haematococca (NhGH11) at 1.0 Å resolution and its functional properties. The atomic resolution structure provides details and insights about the complex hydrogen bonding network of the active site region and allowed a detailed comparison with homologous structures. Complementary biochemical studies showed that the xylanase can catalyze the hydrolysis of complex xylan into simple xylose aldopentose subunits of different lengths. NhGH11 can catalyze the efficient breakdown of beechwood xylan, xylan polysaccharide, and wheat arabinoxylan with turnover numbers of 1730.6 ± 318.1 min-1, 1648.2 ± 249.3 min-1 and 2410.8 ± 517.5 min-1 respectively. NhGH11 showed maximum catalytic activity at pH 6.0 and 45 °C. The mesophilic character of NhGH11 can be explained by distinct structural features in comparison to thermophilic GH11 enzymes, including the number of hydrogen bonds, side chain interactions and number of buried water molecules. The enzymatic activity of NhGH11 is not very sensitive to metal ions and chemical reagents that are typically present in associated industrial production processes. The data we present highlights the potential of NhGH11 to be applied in industrial biomass degradation processes.

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	Figure 1. (A) Cartoon plot of NhGH11 with catalytic active residues are shown in sticks. The structure has a jelly-roll fold characteristic for family GH11 enzymes. The linker region, named cord, and the thumb are indicated. A zoom of the active site region is shown (right figure), with catalytic residues surrounded by solvent molecules in the active site cavity. (B) Topology diagram for NhGH11 is shown with the residue numbers for secondary structure elements and the location of the catalytically essential residues E89 and E180 is indicated.
	Figure 2. (A) Sequence alignment and comparison with homologous GH11 enzymes. Secondary structure regions are indicated along with gray stars indicating residues with alternative side chain conformations in the NhGH11 structure. Sequence alignment was done applying ESPript3 (http://espript.ibcp.fr/ESPript/ESPript). Residues in red color are identical and residues in pink color show similarity among aligned sequences. Catalytic residues E89 and E180 are indicated with filled circles. For the structure with pdb code 4HK8 the second catalytic E180 was mutated to Q180 for ligand binding studies (B) Stereo view of the surface representation with under laid Cα tracing. NhGH11 superimposed with homologue structures (pdb codes: 5JRM, 2JIC, 4HK8, 1H1A, 1YNA and 1PVX) from F. oxysporum, T. longibrachiatum, H. jecorina, C. thermophilum, T. lanuginosus and B. spectabilis respectively. Backbone in red color indicates regions with sequence identity for all structures, the highly conserved thumb region is indicated by black arrows.
	Figure 3. (A) Cartoon figure with residues highlighted in stick mode lining the active site cleft and forming hydrogen bonds with solvent molecules. (B) Catalytic residues E89 and E180 are forming hydrogen bonds with solvent molecules and surrounding residues. The positions of clearly visible hydrogen atoms in active site cleft are indicated by black arrows. 2Fo-Fc electron density maps are shown at 1.0σ level and all shown hydrogen bonds have distances of ~ 3.0 Å.
	Figure 4. Circular Dichroism (CD) spectra at different temperatures recorded in the range of + 20 to + 90 °C, measurements were done with 10 °C intervals.
	Figure 5. (A) Stereo figure showing buried water molecules with electron density within in the cartoon surface representation. 2Fo-Fc electron density maps for water molecules and surrounding residues are shown at 1.0σ level. (B) Selected intramolecular aromatic-aromatic (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \uppi{\cdots}\uppi $$\end{document}π⋯π) interactions and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\text{C-H}}{\cdots} \uppi $$\end{document}C-H⋯π interactions. Interactions were considered if the distance between the interacting proton and centroid of the phenyl group is less than 3.5 Å.
	Figure 6. Kinetics with different substrates (A) Activity with beechwood xylan (B) Activity with xylan polysaccharide (C) Activity with wheat arabinoxylan.

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Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed