Crystal structure of an inverting GH 43 1,5-α-L-arabinanase from Geobacillus stearothermophilus complexed with its substrate

Arabinanases are glycosidases that hydrolyse alpha-(1 -> 5)-arabinofuranosidic linkages found in the backbone of the pectic polysaccharide arabinan. Here we describe the biochemical characterization and the enzyme-substrate crystal structure of an inverting family 43 arabinanase from Geobacillus stearothermophilus T-6 (AbnB). Based on viscosity and reducing power measurements, and based on product analysis for the hydrolysis of linear arabinan by AbnB, the enzyme works in ail endo mode of action. Isothermal titration calorimetry studies of a catalytic mutant with various arabino-oligosaccharides suggested that the enzyme active site can accommodate at least live arabinose units. The crystal structure of AbnB was determined at 1.06 angstrom (1 angstrom = 0.1 nm) resolution, revealing a single five-bladed-beta-propeller fold domain. Co-crystallization of catalytic mutants of the enzyme with different substrates allowed us to obtain complex structures of AbnBE201A with arabinotriose and AbnBD147A with arabinobiose. Based oil the crystal structures of AbnB together with its substrates, the position of the three catalytic carboxylates: Asp(27), the general base; Glu(201), the general acid: and Asp(147), the pK(a) modulator, is in agreement with their putative catalytic roles. In the complex structure of AbnBE201A with arabinotriose, a single water molecule is located 2.8 angstrom from Asp(27) and 3.7 angstrom from the anomeric carbon. The position of this water molecule is kept via hydrogen building with a conserved tyrosine (Tyr(229)) that is 2.6 angstrom distant from it. The location of this molecule suggests that it call function as the catalytic water molecule ill the hydrolysis reaction, resulting in the inversion of the anomeric configuration of the product.