Glycoside Hydrolase Family 119

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• Author: Eduardo Moreno Prieto
• Responsible Curators: Stefan Janecek and Bernard Henrissat

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Glycoside Hydrolase Family GH119
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	known/not known
CAZy DB link
https://www.cazy.org/GH119.html

Substrate specificities

Glycoside hydrolase family 119 (GH119) contains a relatively small number of exclusively bacterial sequences. The first experimentally-characterized member was the amylolytic enzyme IgtZ from Niallia circulans AM7 (formerly Bacillus circulans). Watanabe et al. expressed IgtZ’s full sequence and assayed it against a range of oligo- and polymeric ⍺-glucans, demonstrating its ability to break down amylose and soluble starch but not pullulan or dextran. [1] This suggests a strict specificity towards ⍺-1,4 over ⍺-1,6-glucan bonds. The enzyme also acts on maltooligosaccharides with a minimum degree of polymerization (DP) of 4 and maltooligosyl trehaloses with maltooligosaccharide portions of at least 4 monosaccharide units. The enzyme hydrolytic action yields primarily disaccharides (maltose or trehalose) accompanied by smaller amounts of glucose and other maltodextrins. The enzyme specificity increases with the DP of the substrate. Based on these findings, the authors categorized IgtZ as an ⍺-amylase.[1]

In the CAZy database, the two largest amylolytic families, GH13 and GH57, are notably multi-specific, with ⍺-amylase representing just one of more than 30 specificities in GH13.[2] Family GH119 was predicted in 2012[3] to share its catalytic domain fold and catalytic machinery with those of the family GH57 (Fig. 1). It had been also predicted that GH119 would be largely mono-specific, with ⍺-amylase being the primary specificity.[4] This was based on the composition of one of its conserved sequence regions (CSRs), specifically CSR-5, which correlates with substrate specificity in families GH57 and GH119.[5,6]

Vuillemin et al. expressed recombinantly the core domain of five other GH119 sequences representing the major phylogenetic clades of family GH119.[7] They all showed a very similar specificity as IgtZ’s: activity on glycogen, soluble starch, amylose and maltooligosaccharides with minimum DP5, but not on pullulan or dextran. This result confirmed the in silico prediction of uniform specificity. Their product profiles were also similar to IgtZ’s, except for that of ⍺-amylase CocoGH119 from Corallococcus coralloides DSM 2259, which only produced DP2 and DP3 maltooligosaccharides from longer-chain substrates. This suggests that this enzyme is a maltogenic ⍺-amylase.[7]

Kinetics and Mechanism

Niallia circulans IgtZ is a retaining enzyme as Watanabe et al. confirmed by polarimetry in 2006.[1] The authors did not discuss the enzyme’s possible exo- or endo-acting mode of action.

Catalytic Residues

In silico studies have revealed a close phylogenetic relationship between GH families 119 and 57.[3,4] Through multiple sequence alignment (MSA) and superimposition of GH119 homology models and GH57 crystallographic structures, it has been demonstrated that GH119 sequences share the same five CSRs typical of GH57.[3] The strictly conserved, catalytic residues of GH57, namely a strand β4 glutamate serving as nucleophile and a β7 aspartate serving as acid/base[8,9] are located in CSR-3 and CSR-4, respectively. The equivalent residues in CocoGH119 (E225 and D369) have been found to be essential to catalysis by site-directed mutagenesis experiments resulting in complete abolishment of the enzyme’s activity.[7] Furthermore, homology models of GH119 sequences suggest that their putative catalytic domain may adopt a (β/α)₇-barrel (i.e., an incomplete TIM-barrel) fold followed by a bundle of α-helices.[3,4] These characteristics differentiate the amylolytic families GH57 and GH119 from the main α-amylase family, GH13, which adopts a (β/α)₈-barrel fold (i.e., a classical TIM-barrel) and has a Asp-Glu-Asp catalytic triad, as also observed in GH70 and GH77, all of which are members of clan GH-H.[2] Based on these differences, GH119 and GH57 comprise clan GH-S.[7]

Three-dimensional structures

No 3D structure of a GH119 protein has been experimentally established so far. However, domain prediction indicates that several GH119 proteins exhibit a multi-modular architecture consisting of an N-terminal, putatively-catalytic, main domain followed by a variable number of additional domains, including carbohydrate-binding modules (CBM) of families 20, 25 and 26, fibronectin type III (FN-III) and dockerin domains, among others.[4,7,10] A phylogenetic tree of the family annotated with predicted domain architecture (Fig. 2) shows that different branches exhibit distinctive auxiliary domain patterns.[7] Domain prediction for IgtZ suggests that this enzyme comprises an N-terminal, putative catalytic domain, followed by an FN-III, two CBM20 in tandem and a C-terminal CBM25.[4]

Family Firsts

First stereochemistry determination

The first evidence of a retaining mechanism within the family was presented by Watanabe and colleagues, who conducted a polarimetry study of the maltooligosaccharide products resulting from the hydrolysis of maltopentaosyl trehalose by IgtZ.[1]

First catalytic nucleophile identification

Inferred to be a strictly conserved, strand β4 glutamate located in the CSR-3 of GH119 and GH57 MSAs.[3,4] The equivalent residue in CocoGH119 (E225) is essential to catalysis as demonstrated through site-directed mutagenesis.[7]

First general acid/base residue identification

As above, also inferred through alignment[3,4] and confirmed by site-directed mutagenesis to be a fully conserved, β7 aspartate located in CSR-4 of the family (D369 in CocoGH119).[5]

First 3-D structure

Not yet determined.

References

1. issn=1347-6947

3. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, and Henrissat B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009;37(Database issue):D233-8. DOI:10.1093/nar/gkn663 |

4. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. The Biochemist, vol. 30, no. 4., pp. 26-32. DOI:10.1042/BIO03004026.