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

Abstract

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.

Whole-Genome Sequencing and Comparative Genome Analysis of Fusarium solani-melongenae Causing Fusarium Root and Stem Rot in Sweetpotatoes

“…S5) showed that FsmGH11.3 had the highest sequence similarity (44.1%) with NhGH11 (GenBank accession no. XP_003050975.1 ), a β-1,4-xylanase from GH11 in F. vanettenii was proven to decompose various xylan substrates ( 29 ). This implied that FsmGH11.3 and NhGH11 may share similar enzymatic hydrolysis of xylan.…”

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive

“…GH11 xylanase. N. haematococca GH11 xylanase (GH11) was produced and purified as described previously (Andaleeb et al, 2020). For crystallization, the original conditions were modified to obtain microcrystals.…”

“…For GH11, we processed data with CrystFEL as described above, with the addition that during the partialator step we used both the 'unity' and the 'xsphere' partiality models for comparative reasons. The structure of GH11 (PDB entry 6y0h; Andaleeb et al, 2020) obtained under cryogenic conditions was used as the initial model and refinement was carried out essentially as described above, but without TLS-refinement. For the detailed investigation of the relation of data quality and length of data collection/indexed patterns, phenix.refine was used with exactly the same parameters and without manual intervention.…”

“…This setup has been shown previously to enable efficient SSX experiments using lysozyme as a model system (Beyerlein et al, 2017). We show here the versatility of this setup and methodology by performing structural studies at room temperature (RT) of three additional enzyme systems of biological relevance: (i) Klebsiella pneumoniae CTX-M-14 -lactamase (Wiedorn et al, 2018), a -lactamase relevant to multi-antibiotic resistance mechanisms; (ii) Nectria haematococca xylanase GH11 (Andaleeb et al, 2020), a highly active xylanase suitable for industrial Schematic of the TapeDrive setup as used in this study. The sample capillary can be used with and without mixing, the installation remains the same.…”

LifeSoaks: a tool for analyzing solvent channels in protein crystals and obstacles for soaking experiments

“…In recently published work, the GC11 xylanase from Nectria haematococca (NhGC11) has been investigated with regard to substrate binding and known inhibitors (Andaleeb, 2021). However, for all compounds, soaking a monoclinic crystal of NhGC11 (Andaleeb et al, 2020) did not lead to electron density, hinting at the presence of a ligand in its known binding site. The author attributed this to the dense packing of the monoclinic crystal structure with a low Matthews coefficient of 1.9 A ˚3 Da À1 and narrow solvent channels.…”