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

Yueling Zhang,Jiansong Ju,Hao Peng,Feng Gao,Cheng Zhou,Yan Zeng,Yanfen Xue,Yin Li,Bernard Henrissat,George F Gao,Yanhe Ma

doi:10.1074/jbc.m803409200

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.

Highlights

Endo-␤-1,4-mannanases 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 [5,6,7,8]
We present details pertaining to gene cloning, biochemical characterization, and crystal structure resolution of this novel mannanase
The data strongly suggest that family GH113 belongs to clan glycoside hydrolase (GH)-A. This is the first report on the presence of an endo-␤-1,4-mannanase in the cytoplasm of the thermoacidophilic bacterium A. acidocaldarius

Summary

EXPERIMENTAL PROCEDURES

Substrates and Chemicals—Manno-oligosaccharides (mannobiose, mannotriose, mannotetraose, mannopentose, and mannohexose), ivory nut mannan, yeast mannan, pectic potato galactan, and potato galactan were purchased from Megazyme (Wicklow, Ireland). The substrate concentration was 0.5% (w/v) in buffer A, and incubation was carried out at 65 °C for 10 min with 0.05, 0.1, 5, or 10 ␮M enzyme. Enzymatic activity was quantified by measuring the release of reducing sugars, as described earlier [41]. The plates were incubated at 60 °C for 180 min, and the enzyme activity, which was detected after flooding with 0.1% Congo Red solution, manifested as a clear halo surrounding the “enzyme spot.”. To detect the exoglycosidase activity, the p-nitrophenylglycoside substrate (5 mM) was incubated with 1 ␮M enzyme for 30 min. Time Course of Hydrolysis—The time course of hydrolysis was established using 10 ␮M enzyme in buffer A with 2% (w/v) substrate supplemented with 0.1 mg/ml bovine serum albumin at 60 °C.

Diffraction data and refinement quality statistics for native AaManA

RESULTS AND DISCUSSION

Ivory nut mannan

Substrate kcat

CONCLUSION