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Only a single glycoside hydrolase of family GH113 has been characterized, intracellular AaManA from Alicyclobacillus acidocaldarius Tc-12-31 [1]. This thermoacidophilic organism was originally selected for its ability to hydrolyse konjac glucomannan [1]. The recombinantly-expressed enzyme possesses activity against polysaccharides containing β-1,4-mannosidic linkages, including significant activity against konjac glucomannan, and galactomannan from locust bean gum. Some activity was also observed against crystalline ivory nut mannan (an unsubstituted β-1,4-mannan) and guar gum (a more highly-substituted galactomannan) [1]. No activity was observed against the other polysaccharides and p-nitrophenyl glycosides tested, including p-nitrophenyl β- and α-mannosides.
Kinetics and Mechanism
Thin layer chromatographic analysis of the hydrolysis products of AaManA from A. acidocaldarius indicated endo-type cleavage [1]. The products also displayed obvious signs of transglycosylation, and thus AaManA was assigned a retaining mechanism. The distance between the acid/base and nucleophile residues (assigned on the basis of structural comparison and mutagenesis, see below) is 4.75 Å [1]. Together, these data support a classical Koshland retaining mechanism. Kinetic analysis of mannooligosaccharides reveals a preference for pentamannosides and higher; a tetramannoside was hydrolysed with kcat/KM value approximately one quarter of that seen for the higher oligomers. Crystallographic evidence from a binary complexes of AaManA with β-mannosyl-1,4-mannoimidazole supports a 1S5→B2,5‡→OS2 conformational reaction coordinate [2].
Catalytic Residues
Structural comparision of AaManA with GH5 endoglycosidases demonstrated conserved spatial arrangement of key active site amino acid residues [1]. Structural comparison with Cel5G from Pseudoalteromonas haloplanktis suggested Glu151 to be the general acid/base and Glu231 to be the catalytic nucleophile. The Glu151Ala and Glu231Ala mutants did not affect overall fold but each resulted in a loss of approximately 1000-fold activity against mannan substrates, consistent with the proposed roles. Crystallographic studies with AaManA in complex with β-mannosyl-1,4-mannoimidazole support the assignment of Glu151 as the acid/base [2]. A complex of AaManA with β-mannosyl-1,4-isofagomine supports the assignment of Glu231 as the nucleophile [2].
Three-dimensional structures
The three dimensional structure was first reported for AaManA, which possesses a classical (β/α)8 TIM barrel fold that aligns well with enzymes of family GH5 [1]. Alignment of the AaManA structure with Cel5G from Pseudoalteromonas haloplanktis revealed eight equivalent residues around the proposed active site pocket: Lys93, Thr95, Cys150, Glu151, Ser201, Tyr203, Glu231, and Trp281 [1]. Binary complexes of AaManA with β-mannosyl-1,4-mannoimidazole or β-mannosyl-1,4-isofagomine support the identity of the active site residues originally proposed on the basis of structural comparisons of AaManA with Cel5G and mutagenesis [2]. An active-site spanning ternary complex of AaManA with β-mannosyl-1,4-isofagomine and 1,4-β-mannobiose has provided structural details of amino acids defining the -2 to +2 subsites [2].
