Structures, kinetics, and modeling of glycoside hydrolase family 44 endoglucanases from Clostridium acetobutylicum and Ruminococcus flavefaciens FD-1

Warner, Christopher
Major Professor
Peter J. Reilly
Committee Member
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Chemical and Biological Engineering

Glycoside hydrolases (GH) are enzymes that hydrolyze glycosidic bonds between carbohydrates. Family 44 contains endo- and xylo- glucanases capable of hydrolyzing cellulose and hemicelluose. Two members produced by Clostridium acetobutylicum and Ruminococcus flavefaciens FD-1 are the subjects of characterization work in this dissertation.

The EG from Clostridium acetobutylicum was characterized both structurally and kinetically. Crystallization and x-ray diffraction to 2.2-y resolution revealed a TIM barrel-like structure with additional ψ-loop and β-sandwich folds. The EG hydrolyzes cellotetraose and larger cellooligosaccharides, as well as carboxymethylcellulose and xylan at approximately the same rates. It converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55yC and an optimal pH of 5.0.

The EG from Ruminococcus flavefaciens FD-1 enzyme's catalytic domain, with and without the CBM, hydrolyzes cellotetraose, cellopentaose, cellohexaose, carboxymethylcellulose (CMC), birchwood and larchwood xylan, lichenan, and Avicel, but not cellobiose, cellotriose, mannan, or pullulan. The presence of the CBM increases catalytic efficiencies on both CMC and birchwood xylan. The products of cellooligosaccharide hydrolysis show both asymmetric cleavage and disproportionate product distributions, indicating the reactions taking place are more complicated than hydrolysis alone.

A reaction model, including hydrolysis and transglycosylation, was designed that describes disproportionation of hydrolysis products of cellopentaose and cellohexaose by the two GH44 EGs produced by C. acetobutylicum and R. flavefaciens FD-1. The model fit the C. acetobutylicum EG data well for the digestions of cellotetraose, cellopentaose, and cellohexaose, while agreeing with the absence of measured transglycosylation products in capillary electrophoresis chromatographs due to cellooligosaccharide insolubility or concentrations near or below the limit of detection.

Characterization of binding constants and thermodynamic properties of the novel CBM associated with the R. flavefaciens catalytic domain and its synergistic effect when the two were linked is described. All constructs bind cellotetraose, cellopentaose, and cellohexaose with increasing affinity. No binding of xylotetraose, xylopentaose or xylohexaose was observed. The CBM and fusion protein have larger association constants for CMC than for larchwood xylan. A synergistic effect was observed with the fusion protein on cellopentaose and cellohexaose. All binding experiments were energetically favorable and enthalpy-driven.