title: The GH51 α-l-arabinofuranosidase from Paenibacillus sp. THS1 is multifunctional, hydrolyzing main-chain and side-chain glycosidic bonds in heteroxylans.
abstract: 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 α-l-arabinofuranosidase and β-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 α-l-arabinofuranosidase. Characterization revealed that THSAbf is active up to 75 °C, stable at 60 °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 α-l-arabinofuranosidase, THSAbf also displays endo-xylanase activity, cleaving β-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 α-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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: UBMB, Université de Tunis El Manar, BP 94, 1068 Rommana, Tunisia.|Laboratoire des Ressources Sylvo-Pastorales, Institut Sylvo-Pastoral de Tabarka, Institution de la Recherche et de l'Enseignement Supérieur Agricoles, Université de Jendouba, Jendouba, Tunisia.
authors.forename: Hanen
authors.initials: H
authors.lastname: Bouraoui
authors.affiliation: CNRS, INRA, INSA, LISBP, Université de Toulouse, Toulouse, France.
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Desrousseaux
authors.affiliation: CNRS, INRA, INSA, LISBP, Université de Toulouse, Toulouse, France.|Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, SY23 3DA Ceredigion UK.
authors.forename: Eleni
authors.initials: E
authors.lastname: Ioannou
authors.affiliation: CNRS, INRA, INSA, LISBP, Université de Toulouse, Toulouse, France.
authors.forename: Pablo
authors.initials: P
authors.lastname: Alvira
authors.affiliation: UBMB, Université de Tunis El Manar, BP 94, 1068 Rommana, Tunisia.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Manaï
authors.affiliation: INRA, FARE, Université de Reims Champagne Ardenne, 2, Esplanade Roland Garros, 51100 Reims, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Rémond
authors.affiliation: CNRS, INRA, INSA, LISBP, Université de Toulouse, Toulouse, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Dumon
authors.affiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, SY23 3DA Ceredigion UK.
authors.forename: Narcis
authors.initials: N
authors.lastname: Fernandez-Fuentes
authors.affiliation: CNRS, INRA, INSA, LISBP, Université de Toulouse, Toulouse, France.
authors.forename: Michael J
authors.identifier: 0000-0003-4246-3938
authors.initials: MJ
authors.lastname: O'Donohue
keywords: Biomass; Enzyme cocktails; Glycoside hydrolase; Wheat bran; Xylanase