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Glycoside Hydrolase Family 1

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Glycoside Hydrolase Family GH1
Clan GH-A
Mechanism retaining
Active site residues known
CAZy DB link

Substrate specificities

The most common known enzymatic activities for glycoside hydrolases in this family are β-glucosidases and β-galactosidases: indeed typically both activities are found within the same active site, often with similar kcat values, but with substantially higher Km values for the galactosides. However, other commonly found activities are 6-phospho-β-glucosidase and 6-phospho-β-galactosidase, β-mannosidase, β-D-fucosidase and β-glucuronidase. Family GH1 enzymes are found across a broad spectrum of life forms. Enzymes of medical interest include the human lactase/phlorizin hydrolase whose deficiency leads to lactose intolerance. In plants Family GH1 enzymes are often involved in the processing of glycosylated aromatics such as saponins and some plant hormones stored in inactive glycosylated forms. Indeed some have been identified as plant oncogenes due to aberrant control of auxin levels. Some plants also use Family GH1 enzymes as part of their defense system in order to release toxic aglycons, the most known examples being Trifolium repens β-glucosidase and Sinapis alba myrosinase, which respectively hydrolyse linamarin and glucosinolates. One of the work horses of glycosidase enzymology, the almond emulsin β-glucosidase, even though not fully sequenced, is deduced to belong to Family GH1 by limited sequence analysis [1].

Kinetics and Mechanism

Family GH1 β-glycosidases are retaining enzymes, as first shown by NMR [2] and follow a classical Koshland double-displacement mechanism. Enzymes that have been well-studied kinetically include the almond emulsin enzyme, for which a particularly nice and important set of studies on rate-limiting steps and inhibition was reported in the mid 1980’s [3, 4] and the Agrobacterium sp. β-glucosidase which has been the subject of a series of kinetic evaluations, including detailed steady state [5, 6] and pre-steady state kinetic analyses in which the roles of each substrate hydroxyl in catalysis have also been carefully probed [7].

Catalytic Residues

The catalytic nucleophile was first identified in the Agrobacterium sp. β-glucosidase as Glu358 in the sequence YITENG through trapping of the 2-deoxy-2-fluoroglucosyl-enzyme intermediate and subsequent peptide mapping [8]. The general acid/base catalyst was first identified as Glu170 in this same enzyme through detailed mechanistic analysis of mutants at that position, which included azide rescue experiments [9]. Family GH1 enzymes, as is typical of Clan GH-A, have an asparagine residue preceding the general acid/base catalyst in a typical NEP sequence. The asparagine engages in important hydrogen bonding interactions with the substrate 2-hydroxyl. Interestingly, the plant myrosinases cleave thioglycosides bearing an anionic aglycone (glucosinolates), and have evolved an active site in which the acid/base glutamate is replaced by glutamine. Substrates are sufficiently reactive not to require the acid catalyst, while the role of base catalyst is played by exogenous ascorbate, which binds to the glycosyl enzyme [10]. For a related example see the mannose-1-phosphate-6-mannoside cleaving α-mannosidases of family GH92, which also lack an enzymic general acid residue.

Three-dimensional structures

Three-dimensional structures are available for a large number of Family 1 enzymes, the first solved being that of the white clover (Trifolium repens) cyanogenic β-glucosidase [11]. As members of Clan GH-A they have a classical (α/β)8 TIM barrel fold with the two key active site glutamic acids being approximately 200 residues apart in sequence and located at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) [12].

"Family Firsts"

First sterochemistry determination
Agrobacterium sp. (formerly Alcaligenes faecalis) β-glucosidase by NMR [2]
First catalytic nucleophile identification
Agrobacterium sp. (formerly Alcaligenes faecalis) β-glucosidase by 2-fluoroglucose labeling [8]
First general acid/base residue identification
Agrobacterium sp. (formerly Alcaligenes faecalis) β-glucosidase by rescue kinetics with mutants [9]
First 3-D structure of a GH1 enzyme
White clover (Trifolium repens) cyanogenic β-glucosidase [11]


  1. He S and Withers SG. (1997). Assignment of sweet almond beta-glucosidase as a family 1 glycosidase and identification of its active site nucleophile. J Biol Chem. 1997;272(40):24864-7. DOI:10.1074/jbc.272.40.24864 | PubMed ID:9312086 [1]
  2. Withers SG, Dombroski D, Berven LA, Kilburn DG, Miller RC Jr, Warren RA, and Gilkes NR. (1986). Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex. Biochem Biophys Res Commun. 1986;139(2):487-94. DOI:10.1016/s0006-291x(86)80017-1 | PubMed ID:3094517 [2]
  3. Dale MP, Ensley HE, Kern K, Sastry KA, and Byers LD. (1985). Reversible inhibitors of beta-glucosidase. Biochemistry. 1985;24(14):3530-9. DOI:10.1021/bi00335a022 | PubMed ID:3929833 [3]
  4. Dale MP, Kopfler WP, Chait I, and Byers LD. (1986). Beta-glucosidase: substrate, solvent, and viscosity variation as probes of the rate-limiting steps. Biochemistry. 1986;25(9):2522-9. DOI:10.1021/bi00357a036 | PubMed ID:3087421 [4]
  5. Kempton JB and Withers SG. (1992). Mechanism of Agrobacterium beta-glucosidase: kinetic studies. Biochemistry. 1992;31(41):9961-9. DOI:10.1021/bi00156a015 | PubMed ID:1390780 [5]
  6. Hyttel J (1977). Effect of a selective 5-HT uptake inhibitor--Lu 10-171--on rat brain 5-HT turnover. Acta Pharmacol Toxicol (Copenh). 1977;40(3):439-46. | Google Books | Open Library PubMed ID:139078 [6]
  7. Namchuk MN and Withers SG. (1995). Mechanism of Agrobacterium beta-glucosidase: kinetic analysis of the role of noncovalent enzyme/substrate interactions. Biochemistry. 1995;34(49):16194-202. DOI:10.1021/bi00049a035 | PubMed ID:8519777 [7]
  8. Withers, S. G.; Warren, R. A. J.; Street, I. P.; Rupitz, K.; Kempton, J. B.; Aebersold, R. Journal of the American Chemical Society '1990', 112, 5887-5889.

  9. Wang Q, Trimbur D, Graham R, Warren RA, and Withers SG. (1995). Identification of the acid/base catalyst in Agrobacterium faecalis beta-glucosidase by kinetic analysis of mutants. Biochemistry. 1995;34(44):14554-62. DOI:10.1021/bi00044a034 | PubMed ID:7578061 [9]
  10. Burmeister WP, Cottaz S, Rollin P, Vasella A, and Henrissat B. (2000). High resolution X-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base. J Biol Chem. 2000;275(50):39385-93. DOI:10.1074/jbc.M006796200 | PubMed ID:10978344 [13]
  11. Barrett T, Suresh CG, Tolley SP, Dodson EJ, and Hughes MA. (1995). The crystal structure of a cyanogenic beta-glucosidase from white clover, a family 1 glycosyl hydrolase. Structure. 1995;3(9):951-60. DOI:10.1016/s0969-2126(01)00229-5 | PubMed ID:8535788 [12]
  12. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, and Davies G. (1995). Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1995;92(15):7090-4. DOI:10.1073/pnas.92.15.7090 | PubMed ID:7624375 [10]

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