The GH51 α-L-arabinofuranosidase from Paenibacillus sp THS1 is multifunctional, hydrolyzing main-chain and side-chain glycosidic bonds in heteroxylans

Background: Conceptually, multi-functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity. Nevertheless, multi-functional enzymes are quite rare and are generally multi-domain assemblies with each activity being defined by a separate protein module. However, a recent report described a GH51 arabinofuranosidase from Alicyclobacillus sp. A4 that displays both a-l-arabinofuranosidase and beta-D-xylanase activities, which are defined by a single active site. Following on from this, we describe in detail another multi-functional GH51 arabinofuranosidase and discuss the molecular basis of multifunctionality. Results: THSAbf is a GH51 alpha-L-arabinofuranosidase. Characterization revealed that THSAbf is active up to 75 degrees C, stable at 60 degrees C and active over a broad pH range (4-7). THSAbf preferentially releases para-nitrophenyl from the L-arabinofuranoside (k(cat)/K-M = 1050 s(-1) mM(-1)) and to some extent from D-galactofuranoside and D-xyloside. THSAbf is active on 4-O-methylglucuronoxylans from birch and beechwood (10.8 and 14.4 U mg(-1), respectively) and on sugar beet branched and linear arabinans (1.1 +/- 0.24 and 1.8 +/- 0.1 U mg(-1)). Further investigation revealed that like the Alicyclobacillus sp. A4 alpha-L-arabinofuranosidase, THSAbf also displays endo-xylanase activity, cleaving beta-1,4 bonds in heteroxylans. The optimum pH for THASAbf activity is substrate dependent, but ablation of the catalytic nucleophile caused a general loss of activity, indicating the involvement of a single active center. Combining the alpha-L-arabinofuranosidase with a GH11 endoxylanase did not procure synergy. The molecular modeling of THSAbf revealed a wide active site cleft and clues to explain multi-functionality. Conclusion: The discovery of single active site, multifunctional enzymes such as THSAbf opens up exciting avenues for enzyme engineering and the development of new biomass-degrading cocktails that could considerably reduce enzyme production costs.