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

Abstract

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 ...

A Novel Glycoside Hydrolase Family 113 Endo-β-1,4-Mannanase from Alicyclobacillus sp. Strain A4 and Insight into the Substrate Recognition and Catalytic Mechanism of This Family

“…When using galactomannans with increased galactose contents of 21, 28, 34, and 38% as the substrates, Man113A showed decreased activities of 391.3, 302.8, 36.2, and 34.9 U/mg, respectively. In comparison to its counterparts, Man113A exhibited much lower activity than that of AaManA (1,056 U/mg) toward konjac flour (7) and that of MAN5A from Bispora sp. strain MEY-1 (3,373 U/mg) against locust bean gum (15) but higher activity than that of Man5XZ3 from Aspergillus nidulans XZ3 (184.8 U/mg against locust bean gum) (16).…”

“…By means of molecular docking and experimental confirmation, Man113A was proved to possess the eight functionally conserved residues of GH clan A, as AaManA does (7). To date, microbial ␤-mannanases are classed into GH families 5, 26, and 113, all belonging to GH clan A.…”

“…The docking grid, with an appropriate size of 40 Å by 40 Å by 40 Å, was set to center the ␤-carbon atom of Glu144, which was predicted to be the nucleophile of Man113A. The docked complexes were analyzed based on previous studies (7,13) to select the most reliable binding conformation. Three-dimensional molecular visualization and figure preparation were performed with PyMOL (version 1.7.2.1; Delano Scientific, USA).…”

“…Two glutamate residues at the C termini of ␤ 4 and ␤ 7 sheets serve as a general acid/base and a nucleophile to catalyze the cleavage of glycosidic bonds via a retaining double-displacement mechanism. As yet, 180 sequences have been classified into GH 113 (http://www.cazy.org/GH113 .html); moreover, only one member, AaManA from Alicyclobacillus acidocaldarius Tc-12-31, has been functionally characterized and its structure resolved (7). AaManA is an intracellular endo-␤-1,4-mannanase with transglycosylation activity, and several residues related to substrate binding are identified by sequence alignments and structure analysis.…”

Unveiling the beauty of Archaea

“…Their research covers chromosomal organization, genetic mechanisms, viruses and plasmids, synthesis of polyhydroxyalkanoates (PHAs), quorum sensing, enzyme stability, etc. [25][26][27][28][29][30][31][32][33][34][35][36]. Although moderate in size, the Chinese archaeal community has made important contributions to the understanding of Archaea and is now well respected in the field.…”