Seminars Archive

Structural and functional studies of Bacillus pumilus acetyl xylan esterase: Insights into the catalytic activity of a hexameric enzymatic assembly

Abstract
Bacillus pumilus PS 213 Acetyl Xylan Esterase (AXE, EC 3.1.1.6) belongs to the Carbohydrate Esterase family seven (CE7). This family groups bacterial enzymes with primary structure similarities displaying the classical α/β hydrolase fold encased in a hexameric assembly. The enzymes show activity towards a broad range of acetylated compounds including short acetylated xylooligosaccharides and the antibiotic Cephalosporin C. B. pumilus AXE was crystallized in a P21 space group and two native crystal forms AXE I and AXE II were characterized at 1.9 Å and 2.5 Å resolution, respectively. Twelve molecules arranged in two doughnut-like 32 hexamers were placed successfully in each asymmetric unit. In order to provide structural details on the working mechanism of this class of hexameric esterases, the crystal structure of AXE in presence of both sodium acetate and xylose, as well as the crystal structure of the binary complex AXE S181A inactive mutant – β -D-xylopyranose, were determined at 2.6 Å. The obtained structural data suggest that an enlargement of the central opening of the hexamer may facilitate the substrate access to the internal chamber and to the enclosed active sites, whereas a putative back-door exit may control the release of small products from each catalytic centre. Snapshots of the catalytic deacylation/acylation process were captured. Structural based description of the acyl-enzyme state, of the tetrahedral reaction intermediate formation and of the acetate leaving the oxyanion hole will be presented. The positional deacetylation specificity of B. pumilus AXE was determined on monoacetylated 4-nitrophenyl b-D-xylopyranosides using the enzyme-coupled assay. The highest affinity was observed for the 4-O-acetyl derivative indicating different deacetylation positional specificity of this enzyme in comparison to the acetyl xylan esterases from the other families that have been tested so far on the mentioned substrates. The unusual inhibition resistance shown by B. pumilus AXE and other members of the CE7 family towards the PMSF could be used as a criterion for intrafamily structural-functional diversification as underlined also by the phylogenetic tree representation of the CE7 members.