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Glycoside Hydrolase Family 15
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|Glycoside Hydrolase Family GH15|
|Active site residues||known|
|CAZy DB link|
Glycoside hydrolases of this family are exo-acting enzymes that hydrolyze the non-reducing end residues of α-glucosides. At present, the most commonly characterized activity is glucoamylase (EC 188.8.131.52), also know as amyloglucosidase, but glucodextranase (EC 184.108.40.206) and α,α-trehalase (EC 220.127.116.11) activities have been described. It has been found that fungal glucoamylases present some substrate flexibility and are able to degrade not only α-1,4-glycosidic bonds but also α-1,6-, α-1,3- and α-1,2-bonds to a lower degree .
Kinetics and Mechanism
Family GH15 α-glycosidases are inverting enzymes, as first shown by Weil et al., 1954  and follow a classical Koshland single-step displacement mechanism. Enzymes that have been well studied kinetically include the Aspergillus and Rhizopus glucoamylases.
The general acid was first identified in the Aspergillus awamori / Aspergillus niger glucoamylase as Glu179 following site-directed mutagenesis . The general base was defined as Glu400 following the three-dimensional structure determination  and confirmed later on by site directed mutagenesis and kinetic studies . Simultaneously the general base was identified in Clostridium sp. G0005 glucoamylase by chemical modification and mutagenesis .
Three-dimensional structures are available for several GH15 family enzymes, the first solved being that of Aspergillus awamori var. X100 glucoamylase . All members of this family have (α/α)6 barrel fold with the two key catalytic glutamic acid residues being approximately 200 residues apart in sequence and located at the loops following barrel α-helices 5 (general acid) and 11 (general base). Bacterial GH15 enzymes have in general an all β-strand super-β-sandwich preceding the catalytic (α/α)6 barrel .
- First sterochemistry determination
- First sequence identification
Aspergillus niger glucoamylase by peptide sequencing .
- First general acid identification
Aspergillus awamori glucoamylase from mutant kinetic analysis .
- First general base identification
Aspergillus awamori var. X100 glucoamylase from crystal structure .
- First 3-D structure
Aspergillus awamori var. X100 glucoamylase by X-ray cristallography .
- Meagher MM and Reilly PJ. (1989) Kinetics of the hydrolysis of di- and trisaccharides with Aspergillus niger glucoamylases I and II. Biotechnol Bioeng. 34, 689-93. DOI:10.1002/bit.260340513 |
- Weil CE, Burch RJ, Van Dyk JW. An α-amyloglucosidase that produces β-glucose, Cereal Chem 1954; 31 150–158.
- Sierks MR, Ford C, Reilly PJ, and Svensson B. (1990) Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamori. Protein Eng. 3, 193-8.
- Harris EM, Aleshin AE, Firsov LM, and Honzatko RB. (1993) Refined structure for the complex of 1-deoxynojirimycin with glucoamylase from Aspergillus awamori var. X100 to 2.4-A resolution. Biochemistry. 32, 1618-26.
- Frandsen TP, Dupont C, Lehmbeck J, Stoffer B, Sierks MR, Honzatko RB, and Svensson B. (1994) Site-directed mutagenesis of the catalytic base glutamic acid 400 in glucoamylase from Aspergillus niger and of tyrosine 48 and glutamine 401, both hydrogen-bonded to the gamma-carboxylate group of glutamic acid 400. Biochemistry. 33, 13808-16.
- Ohnishi H, Matsumoto H, Sakai H, and Ohta T. (1994) Functional roles of Trp337 and Glu632 in Clostridium glucoamylase, as determined by chemical modification, mutagenesis, and the stopped-flow method. J Biol Chem. 269, 3503-10.
- Aleshin A, Golubev A, Firsov LM, and Honzatko RB. (1992) Crystal structure of glucoamylase from Aspergillus awamori var. X100 to 2.2-A resolution. J Biol Chem. 267, 19291-8.
- Aleshin AE, Feng PH, Honzatko RB, and Reilly PJ. (2003) Crystal structure and evolution of a prokaryotic glucoamylase. J Mol Biol. 327, 61-73.
- Svensson S, Larsen K, Svendsen I, Boel E. The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger. Carlsberg Res Commun 1983; 48(6) 529-44 DOI: 10.1007/BF02907555