Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus

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.