Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus

Abstract

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance.

Keywords: CAZymes; cellulose; enzyme synergy; lignocellulosic biomass; multifunctional enzymes; xylan.

Figure 1

(A) Analysis of relative activity of GuxA on Avicel (green), CMC (blue) and xylan (red) at various pH conditions. (B) Analysis of relative activity GuxA on Avicel (green), CMC (blue) and xylan (red) at various temperatures.

Figure 2

Investigation of the synergistic effects of GuxA with E1 on the deconstruction of Avicel using various mass ratios. Digestions were carried out for 72 h.

Figure 3

Investigation of the deconstruction of APCS: Glucan (A) and xylan (B) conversion using single enzymes and the two most extreme loadings of GuxA and E1 from Figure 2. Digestions were carried out for 72 h.

Figure 4

The crystal structure of the GuxA GH12 domain. (A) Viewed perpendicular to the β-jelly roll with β-strands labelled (N.B. strands A6 and B1, and parts of B7-B9 were not rendered as β-strands by PyMol). (B) The GH12 domain rotated 90° from A, viewed down the active site cavity. (C) Features of interest on the cellobiose bound structure—The two disulfides are shown in orange with cellobiose (cyan), the residues that coordinate it (blue) and the catalytic glutamate residues (light red) shown enlarged and reoriented in panel (D).