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Glycoside Hydrolase Family 42

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Glycoside Hydrolase Family GH42
Clan GH-A
Mechanism retaining
Active site residues known
CAZy DB link

Substrate specificities

The most common activity for glycoside hydrolases of this family are β-galactosidases (EC, however, other commonly found activities are α-L-arabinosidase (EC and β-D-fucosidase (EC 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 [6]. 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 [9], and with cellulosome [1].

The activity of GH42 enzymes on lactose and also lactulose [2] 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 [10], 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.

Catalytic Residues

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 [11]. 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 [2].

Three-dimensional structures

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 [10]. 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.

Family Firsts

First stereochemistry determination
T. thermophilus A4 β-galactosidase by NMR [10].
First catalytic nucleophile identification
B. subtilis YesZ β-galactosidase by 2-fluorogalactose labeling [11].
First general acid/base residue identification
A. acidocaldarius β-galactosidase by rescue kinetics with mutant [2].
First 3-D structure
T. thermophilus A4 β-galactosidase [10].


  1. Kosugi A, Murashima K, and Doi RH. (2002). Characterization of two noncellulosomal subunits, ArfA and BgaA, from Clostridium cellulovorans that cooperate with the cellulosome in plant cell wall degradation. J Bacteriol. 2002;184(24):6859-65. DOI:10.1128/JB.184.24.6859-6865.2002 | PubMed ID:12446636 [1]
  2. Di Lauro B, Strazzulli A, Perugino G, La Cara F, Bedini E, Corsaro MM, Rossi M, and Moracci M. (2008). Isolation and characterization of a new family 42 beta-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius: identification of the active site residues. Biochim Biophys Acta. 2008;1784(2):292-301. DOI:10.1016/j.bbapap.2007.10.013 | PubMed ID:18068682 [2]
  3. Ohtsu N, Motoshima H, Goto K, Tsukasaki F, and Matsuzawa H. (1998). Thermostable beta-galactosidase from an extreme thermophile, Thermus sp. A4: enzyme purification and characterization, and gene cloning and sequencing. Biosci Biotechnol Biochem. 1998;62(8):1539-45. DOI:10.1271/bbb.62.1539 | PubMed ID:9757561 [3]
  4. Kang SK, Cho KK, Ahn JK, Bok JD, Kang SH, Woo JH, Lee HG, You SK, and Choi YJ. (2005). Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization. J Biotechnol. 2005;116(4):337-46. DOI:10.1016/j.jbiotec.2004.07.019 | PubMed ID:15748760 [4]
  5. Yuan T, Yang P, Wang Y, Meng K, Luo H, Zhang W, Wu N, Fan Y, and Yao B. (2008). Heterologous expression of a gene encoding a thermostable beta-galactosidase from Alicyclobacillus acidocaldarius. Biotechnol Lett. 2008;30(2):343-8. DOI:10.1007/s10529-007-9551-y | PubMed ID:17914606 [5]
  6. Møller PL, Jørgensen F, Hansen OC, Madsen SM, and Stougaard P. (2001). Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization. Appl Environ Microbiol. 2001;67(5):2276-83. DOI:10.1128/AEM.67.5.2276-2283.2001 | PubMed ID:11319112 [6]
  7. Van Laere KM, Abee T, Schols HA, Beldman G, and Voragen AG. (2000). Characterization of a novel beta-galactosidase from Bifidobacterium adolescentis DSM 20083 active towards transgalactooligosaccharides. Appl Environ Microbiol. 2000;66(4):1379-84. DOI:10.1128/AEM.66.4.1379-1384.2000 | PubMed ID:10742215 [7]
  8. Hinz SW, van den Brock LA, Beldman G, Vincken JP, and Voragen AG. (2004). beta-galactosidase from Bifidobacterium adolescentis DSM20083 prefers beta(1,4)-galactosides over lactose. Appl Microbiol Biotechnol. 2004;66(3):276-84. DOI:10.1007/s00253-004-1745-9 | PubMed ID:15480628 [8]
  9. Shipkowski S and Brenchley JE. (2006). Bioinformatic, genetic, and biochemical evidence that some glycoside hydrolase family 42 beta-galactosidases are arabinogalactan type I oligomer hydrolases. Appl Environ Microbiol. 2006;72(12):7730-8. DOI:10.1128/AEM.01306-06 | PubMed ID:17056685 [9]
  10. Hidaka M, Fushinobu S, Ohtsu N, Motoshima H, Matsuzawa H, Shoun H, and Wakagi T. (2002). Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose. J Mol Biol. 2002;322(1):79-91. DOI:10.1016/s0022-2836(02)00746-5 | PubMed ID:12215416 [10]
  11. Shaikh FA, Müllegger J, He S, and Withers SG. (2007). Identification of the catalytic nucleophile in Family 42 beta-galactosidases by intermediate trapping and peptide mapping: YesZ from Bacillus subtilis. FEBS Lett. 2007;581(13):2441-6. DOI:10.1016/j.febslet.2007.04.053 | PubMed ID:17485082 [11]

All Medline abstracts: PubMed