title: 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-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 angstroms resolution showed that AaManA adopted a (beta/alpha)8-barrel fold. Two catalytic residues were identified: Glu151 at the C terminus of beta-stand beta4 and Glu231 at the C terminus of beta7. 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 Thr95 and Cys150. We propose a model of substrate binding in AaManA in which Glu282 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.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Yueling
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jiansong
authors.initials: J
authors.lastname: Ju
authors.forename: Hao
authors.initials: H
authors.lastname: Peng
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhou
authors.forename: Yan
authors.initials: Y
authors.lastname: Zeng
authors.forename: Yanfen
authors.initials: Y
authors.lastname: Xue
authors.forename: Yin
authors.initials: Y
authors.lastname: Li
authors.forename: Bernard
authors.initials: B
authors.lastname: Henrissat
authors.forename: George F
authors.initials: GF
authors.lastname: Gao
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma