Biochemical and Structural Characterization of the Intracellular Mannanase AaManA of Alicyclobacillus acidocaldarius Reveals a Novel Glycoside Hydrolase Family Belonging to Clan GH-A

An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-beta-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9 A resolution showed that AaManA adopted a (beta/alpha)8-barrel fold. Two catalytic residues were identified: Glu(151) at theCterminus of beta-stand beta 4 and Glu(231) at the C terminus of beta 7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr(95) and Cys(150). We propose a model of substrate binding in AaManAin which Glu(282) interacts with the axial OH-C(2) in -2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.