Glycoside Hydrolase Family 113

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Authors: ^^^Rohan Williams^^^ and ^^^Spencer Williams^^^
Responsible Curator: ^^^Spencer Williams^^^

Glycoside Hydrolase Family GH113
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH113.html

Substrate specificities

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 possessed 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 k_cat/K_M value approximately one quarter of that seen for the higher oligomers.

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 ‘’Aa’’ManA 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]. Crystallographic evidence from a binary complexes of AaManA with β-mannosyl-1,4-mannoimidazole supports a ¹’’S’’₅→‘’B’’_2,5^‡→^O’’S’’₂ conformational reaction coordinate [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 ‘’Aa’’ManA 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].

Family Firsts

First stereochemistry determination: AaManA was shown to be retaining by evidence for transglycosylation as well as the spatial separation of the general acid/base and catalytic nucleophile [1].
First catalytic nucleophile identification: Glu231 in AaManA by structural study supported by mutagenesis [1].
First general acid/base residue identification: Glu151 in AaManA by structural study supported by mutagenesis [1].
First 3-D structure: AaManA from A. acidocaldarius, PDB code 3CIV [1] .

References