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Glycoside Hydrolase Family 42
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|Glycoside Hydrolase Family GH42|
|Active site residues||known|
|CAZy DB link|
The most common activity for glycoside hydrolases of this family are β-galactosidases (EC 188.8.131.52), however, other commonly found activities are α-L-arabinosidase (EC 184.108.40.206) and β-D-fucosidase (EC 220.127.116.11) with both KM and kcat values being of the same order of magnitude for the different substrates [1, 2]. Apparently, these enzymes show strict specificity for axial C4-OH groups.
Family GH42 enzymes have been identified only in unicellular organisms, mainly from prokaryotes (in majority bacteria), and with a few examples from archaea and fungi. GH42 enzymes are active on lactose [2, 3, 4, 5] and transgalactosylation was observed with production of galactooligosaccharides . However, several GH42 enzymes are extracted from diverse habitats where lactose would not be present and they are very active on galactooligosaccharides and galactans [1, 7, 8, 9], suggesting that these enzymes would be involved in vivo in plant cell wall degradation. This function could be performed in cooperation with family GH53 galactanases, often encoded from genes adjacent to GH42 genes , and with cellulosome .
The activity of GH42 enzymes on lactose and also lactulose  has interesting potential for the removal of the former from dairy products and to monitor lactulose concentration during heat treatment leading to UHT milk.
Kinetics and Mechanism
Family GH42 β-galactosidase are retaining enzymes, as first shown by NMR , and follow the classical Koshland double-displacement mechanism. Enzymes whose reaction mechanism has been well studied include the β-galactosidase from Thermus thermophilus A4, YesZ from Bacillus subtilis, and the enzyme from Alicyclobacillus acidocaldarius.
The catalytic nucleophile was first identified in the B. subtilis YesZ β-galactosidase as Glu295 through the use of a mechanism-based inhibitor that allowed trapping of the 2-deoxy-2-fluorogalactosyl-enzyme intermediate and subsequent peptide mapping. These experiments were performed on the mutant of the inferred acid/base, which was more sensitive to the inhibitor . The general acid/base catalyst was first identified as Glu157 in the β-galactosidase from A. acidocaldarius through detailed mechanistic analysis and azide rescue experiments of a mutant in that position .
The three-dimensional structure of the β-galactosidase from T. thermophilus A4 was solved at 1.6 Å and 2.2 Å resolution in the free and galactose-bound enzyme, respectively . As members of Clan GH-A, Family GH42 enzymes present the catalytic dyad at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) within a domain showing a classical (α/β)8 TIM barrel (domain A). This catalytic domain contains a metal-binding site with a Zn atom that is not related with the binding of galactose and that thereby seems to have structural features. Domain B, showing a α/β fold domain, is involved in the native trimer formation while the function of domain C (β fold domain) is unknown.
- First stereochemistry determination
- T. thermophilus A4 β-galactosidase by NMR .
- First catalytic nucleophile identification
- B. subtilis YesZ β-galactosidase by 2-fluorogalactose labeling .
- First general acid/base residue identification
- A. acidocaldarius β-galactosidase by rescue kinetics with mutant .
- First 3-D structure
- T. thermophilus A4 β-galactosidase .
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