Glycoside Hydrolase Family 53

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• Author: Leila LoLeggio
• Responsible Curator: Leila LoLeggio

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Glycoside Hydrolase Family GH53
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH53.html

Function and Substrate specificities

The only known specificity for glycoside hydrolases of this family is β-1,4-galactanase (EC 3.2.1.89) and the only reported function is the microbial degradation of galactans and arabinogalactans in the pectic component of plant cell walls. A number of patents on industrial applications of GH53 have been filed. In a number of bacteria, GH53 β-1,4-galactanases genes have been found as part of gene clusters devoted to galactan utilization and additionally comprising genes encoding for a GH42 β-1,4-galactosidase, a galactooligosaccharide transport system and a transcriptional regulator.

Kinetics and Mechanism

GH53 β-1,4-galactanases follow a retaining mechanism as first demonstrated by following the stereochemical course of rection for the endo-β-1,4-galactanase of the bacterium Cellvibrio japonicus (at that time referred to as Pseudomonas fluorescens subspecies cellulosa) [1]. Most characterized members have been reported to be endo-acting, although processivity has been suggested in one case [2].

Catalytic Residues

The catalytic residues were first identified for the endo-β-1,4-galactanase of the bacterium Cellvibrio japonicus [1] (previously known as Pseudomonas fluorescens subspecies cellulosa). Prior to structure determination Henrissat used hydrophobic cluster analysis and sequence alignments to predict that the family belonged to clan GH-A, and the two proposed catalytic residues, which were confirmed by a combination of mutagenesis and kinetic analysis; one acting as a general acid/base (E161) and the other as a catalytic nucleophile (E270).

Three-dimensional structures

As for all members of Clan GH-A [3, 4], structurally characterized GH53 enzymes [5, 6, 7] display a (β/α)₈ barrel structure for the catalytic domain, usually with fairly compact loop structure and a sequence under 400 residues in length. The catalytic residues are typically positioned at the C-terminal ends of βstrands 4 and 7 in the barrel. Somewhat unusually, none of the four structurally characterized GH53 catalytic domains was accompanied by other catalytic domains or accessory modules, but modularity can be inferred by sequence in other members of the family. A disulphide bridging two loops (β/α loops 7 and 8) in 3 known fungal structures [5, 6], is replaced functionally by a calcium ion in one bacterial structure [7]. For one bacterial member of the family ligand complexes with products have been obtained crystallographically, occupying subsites -4 to -2 and +1 to +2 [7, 8]. Based on these crystal structures, binding of a galactononaose fragment has also been computationally modelled [8].

Family Firsts

First stereochemistry determination

Cellvibrio japonicus endo-β-1,4-galactanase [1].

First catalytic nucleophile identification

Cellvibrio japonicus endo-β-1,4-galactanase [1].

First general acid/base residue identification

Cellvibrio japonicus endo-β-1,4-galactanase [1].

First 3-D structure

Aspergillus aculeatus endo-β-1,4-galactanase [5].

References

1. Braithwaite KL, Barna T, Spurway TD, Charnock SJ, Black GW, Hughes N, Lakey JH, Virden R, Hazlewood GP, Henrissat B, and Gilbert HJ. (1997). Evidence that galactanase A from Pseudomonas fluorescens subspecies cellulosa is a retaining family 53 glycosyl hydrolase in which E161 and E270 are the catalytic residues. Biochemistry. 1997;36(49):15489-500. DOI:10.1021/bi9712394 |
2. Hinz SW, Pastink MI, van den Broek LA, Vincken JP, and Voragen AG. (2005). Bifidobacterium longum endogalactanase liberates galactotriose from type I galactans. Appl Environ Microbiol. 2005;71(9):5501-10. DOI:10.1128/AEM.71.9.5501-5510.2005 |
3. Jenkins J, Lo Leggio L, Harris G, and Pickersgill R. (1995). Beta-glucosidase, beta-galactosidase, family A cellulases, family F xylanases and two barley glycanases form a superfamily of enzymes with 8-fold beta/alpha architecture and with two conserved glutamates near the carboxy-terminal ends of beta-strands four and seven. FEBS Lett. 1995;362(3):281-5. DOI:10.1016/0014-5793(95)00252-5 |
4. 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 |
5. Ryttersgaard C, Lo Leggio L, Coutinho PM, Henrissat B, and Larsen S. (2002). Aspergillus aculeatus beta-1,4-galactanase: substrate recognition and relations to other glycoside hydrolases in clan GH-A. Biochemistry. 2002;41(51):15135-43. DOI:10.1021/bi026238c |
6. Le Nours J, Ryttersgaard C, Lo Leggio L, Østergaard PR, Borchert TV, Christensen LL, and Larsen S. (2003). Structure of two fungal beta-1,4-galactanases: searching for the basis for temperature and pH optimum. Protein Sci. 2003;12(6):1195-204. DOI:10.1110/ps.0300103 |
7. Ryttersgaard C, Le Nours J, Lo Leggio L, Jørgensen CT, Christensen LL, Bjørnvad M, and Larsen S. (2004). The structure of endo-beta-1,4-galactanase from Bacillus licheniformis in complex with two oligosaccharide products. J Mol Biol. 2004;341(1):107-17. DOI:10.1016/j.jmb.2004.05.017 |
8. Le Nours J, De Maria L, Welner D, Jørgensen CT, Christensen LL, Borchert TV, Larsen S, and Lo Leggio L. (2009). Investigating the binding of beta-1,4-galactan to Bacillus licheniformis beta-1,4-galactanase by crystallography and computational modeling. Proteins. 2009;75(4):977-89. DOI:10.1002/prot.22310 |