Characterisation of the Effect of the Spatial Organisation of Hemicellulases on the Hydrolysis of Plant Biomass Polymer

Abstract

Synergism between enzymes is of crucial importance in cell metabolism. This synergism occurs often through a spatial organisation favouring proximity and substrate channelling. In this context, we developed a strategy for evaluating the impact of the geometry between two enzymes involved in nature in the recycling of the carbon derived from plant cell wall polymers. By using an innovative covalent association process using two protein fragments, Jo and In, we produced two bi-modular chimeric complexes connecting a xylanase and a xylosidase, involved in the deconstruction of xylose-based plant cell wall polymer. We first show that the intrinsic activity of the individual enzymes was preserved. Small Angle X-rays Scattering (SAXS) analysis of the complexes highlighted two different spatial organisations in solution, affecting both the distance between the enzymes (53 Å and 28 Å) and the distance between the catalytic pockets (94 Å and 75 Å). Reducing sugar and HPAEC-PAD analysis revealed different behaviour regarding the hydrolysis of Beechwood xylan. After 24 h of hydrolysis, one complex was able to release a higher amount of reducing sugar compare to the free enzymes (i.e., 15,640 and 14,549 µM of equivalent xylose, respectively). However, more interestingly, the two complexes were able to release variable percentages of xylooligosaccharides compared to the free enzymes. The structure of the complexes revealed some putative steric hindrance, which impacted both enzymatic efficiency and the product profile. This report shows that controlling the spatial geometry between two enzymes would help to better investigate synergism effect within complex multi-enzymatic machinery and control the final product.

A Comparative Study to Decipher the Structural and Dynamics Determinants Underlying the Activity and Thermal Stability of GH-11 Xylanases

“…Cell lysis was achieved using a 15-mL tube filled with 2 mL of 0.1 mm zirconia-silicate beads (Cole-Parmer™, Vernon Hills, IL, USA) and homogenized for 40 s at 6.5 m s using a FastPrep-24™ 5G (MP Biomedicals, Irvine, CA, USA). Proteins bearing the His -Tag were purified by immobilized metal ion affinity chromatography (IMAC), as previously described [ 43 ].…”

“…The specific activity (SA) of the xylanases was measured using the dinitrosalicylic acid (DNSA) assay, as previously described [ 43 ], in a final concentration of 1% ( ) wheat arabinoxylan (WAX) medium viscosity (Megazyme, Ireland) in 50 mM sodium phosphate, 12 mM sodium citrate pH 6, supplemented with 1 mg/mL of bovine serum albumin (BSA) Sigma. Final concentration of Ev Xyn11 TS and Np Xyn11A is 25 nM and 5 nM, respectively.…”

Effect of multimodularity and spatial organization of glycoside hydrolases on catalysis

“…There is therefore a need to advance the field using an orthogonal "integrative structure determination" approach, integrating structural, biochemical and bioinformatics studies as proposed by Lansky et al [15]. In addition, only a few studies aimed at inducing spatial proximity using protein scaffolds have been developed [56,57]. In these studies, the authors used a covalent association process involving two protein fragments to systematically study spatial organizations within multi-modular GHs, correlating biochemical and structural studies.…”

The covalent complex of Jo-In results from a long-lived, non-covalent intermediate state with near-native structure

“…It consists of two proteins, Jo and In, of similar size (10.5 and 16 kDa, respectively) [9]. This complex has also found some applications in biotechnology, as it allows a simple control on the overall distance between the fused enzymes [10,11].…”