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--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 Å resolution showed that AaManA adopted a (/␣) 8 -barrel fold. Two catalytic residues were identified: Glu 151 at the C terminus of -stand 4 and Glu 231 at the C terminus of 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 AaManA in 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.Mannans are polysaccharides found in plants and consist of a backbone of -1,4-linked mannose and glucose units. Mannose residues often carry an ␣-galactosyl substitute at O-6, and the degree of substitution depends on the plant of origin (Fig. 1A) (1). Mannans are widely distributed in nature in parts of the hemicellulose fraction in hardwoods and softwoods (1), legume seeds (2), and beans of carob trees (3).Endo--1,4-mannanases (mannanases; EC 3.2.1.78) are glycoside hydrolases that randomly cleave the -1,4-linkage in mannans (Fig. 1, A and B) (4); these enzymes have been isolated from bacteria, fungi, plants, and some mollusks (5-8). Interest in these enzymes has been increasing due to their importance in hemicellulose hydrolysis and various other applications (5, 9, 10). During the last 2 decades, more than 80 sequences of the catalytic domains of mannanase have been reported (see the CAZy site on the World Wide Web) and classified into glycoside hydrolase (GH) 2 families 5 and 26, based on their sequence similarities (11). In recent years, crystallographic resolution of the structures of these enzymes has yielded information on their structure. At present, the tertiary structures of seven mannanases have been determined, of which five are from GH family 5 (12-16) and two are from family 26 (17, 18). All of these mannanases share a common (/␣) 8 barrel fold, a retaining reaction mechanism (Fig. 1C), and two conserved catalytic residues (two glutamate residues at the C termini of 4 and 7, respectively). Therefore, all of these enzymes have been assigned to the same GH ...