Arabinan is a branched polysaccharide which 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 is a thermophilic soil bacterium and has an extensive system for the utilization of L-arabinan. The system is composed of 5 transcriptional units and clustered within a 38-kb DNA segment. One of the transcriptional units encodes for 11 genes (abnEFJ-abnA-abfBA-araJKLMN). This operon encodes for arabino-oligosaccharides transporter system, two α-L-arabinofuranosidases (AbfA and AbfB), and a cluster encodes for an alternative arabinose utilization pathway. The last gene in the operon, araN, shares 44% identity (642 amino acid overlap) with a novel β-L- arabinofuranosidase glycoside hydrolase family 127 from Bifidobacterium longum.
Due to the scarcity of β-L-arabinofuranoside residues, neither synthetic nor native substrates are commercially available. However, AraN hydrolyzed pNP-α-D-xylopyranoside, which shares structure similarity with pNP-β-L-arabinofuranoside. Inductive coupled plasma (ICP) measurements showed that AraN contained 3.4 mol of calcium and 0.6 mol of zinc per mol of enzyme. Amino acid sequence analysis revealed three conserved cysteine residues, Cys329, Cys406 and Cys407 that may be involved in metal-binding. Preliminary results showed that a single mutation of Cys406Ala abolished metal binding and affected catalytic activity.
