Glycoside Hydrolase Family 1

Glycoside Hydrolase Family GH1
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH1.html

Substrate specificities

The most common known enzymatic activities for glycoside hydrolases in this family, at the current time, are those of β-glucosidases and β-galactosidases: indeed typically both activities are found within the same active site, often with similar k_cat values, but with substantially higher K_m 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 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 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].

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]

References

All Medline abstracts: PubMed