BIOCHEMICAL CHARACTERIZATION AND CRYSTAL STRUCTURE OF A NOVEL GH127 Β-L- ARABINOFURANOSIDASE

Arabinan is a branched polysaccharide and is part of pectin, one of the components in the plant cell wall. The arabinan backbone consists of α-1,5-linked L-arabinofuranosyl units, and is decorated mainly with α-1,2- and α-1,3-linked arabinofuranosides. Unlike the widespread α configuration of arabinofuranosyl, β-arabinofuranosyl residues are relatively rare in native polysaccharides.

Geobacillus stearothermophilus T-6 is a thermophilic soil bacterium that possesses an extensive system for the utilization of L-arabinan. The system is composed of five transcriptional units, one of which contains eleven genes (abnEFJ-abnA-abfBA-araJKLMN). This operon encodes for an arabino-oligosaccharides transporter system, two α-L-arabinofuranosidases (Abf51A and Abf51B), and several genes encoding for an alternative arabinose utilization pathway. The last gene in the operon, ara127N, encodes for a β-L-arabinofuranosidase and shows activity towards both synthetic and natural substrates containing β-L-arabinofuranoside residues. The k_cat value of Ara127N towards pNP-β-L-arabinofuranoside in pH 6.5 is 1.5 sec^-1, K_m is 0.57 mM and the calculated value of k_cat/ K_m is 2.6 sec^-1·mM^-1. The crystal structure of Ara127N has recently been determined (at 2.30 Å resolution) and it appears to have a metal-binding motif with Zn²⁺ coordinated by glutamate and cysteines in the active site. Inductive coupled plasma (ICP) measurements show that Ara127N contains 0.8 mol of zinc per mol of enzyme. Amino acid sequence analysis revealed three conserved cysteine residues, Cys329, Cys406 and Cys407 that could be involved in metal binding. Preliminary results demonstrated that a single mutation of Cys406Ala abolished metal binding and affected the catalytic activity, suggesting the involvement of the metal in the catalytic mechanism.