Glycoside Hydrolase Family 128

This page has been approved by the Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail.

• Author: ^^^Yuichi Sakamoto^^^ and ^^^Camilla Santos^^^

• Responsible Curator: ^^^Mario Murakami^^^

---

Glycoside Hydrolase Family GH128
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/GH128.html

Substrate specificity and modes of action

The first GH128 enzyme, GLU1, was cloned from Lentinula edodes fruiting bodies (shiitake mushroom) [1]. GLU1 cleaves β-1,3 linkages in various β-glucans such as endogenous L. edodes lentinan, laminarin from Laminaria digitata, pachyman from Poria cocos, and curdlan from Alcaligenes faecalis, but does not degrade β-1,3-linkages within β-1,3-1,4-glucans such as barley glucan, indicating the enzyme is categorized into EC 3.2.1.39 [1]. Further work with several GH128 members corroborated that this family is specific for β-1,3-glucans [2]. Bacterial members from Amycolatopsis mediterranei (subgroup I) and Pseudomonas viridiflava (subgroup II)exhibit endo-β-1,3-glucanase activity and catalytic rates notably higher than those observed for fungal members [2]. On the other hand, fungal members display diverse modes of action and substrate specificity. The GH128 members from Aureobsidium namibiae (subgroup VI) and Cryptococcus neoformans (subgroup V) are exo-β-1,3-glucanases and release trisaccharides and monosaccharides from the reducing ends, respectively. The enzyme from Blastomyces gilchristii (subgroup III) is also an exo-β-1,3-glucanase; however, it releases trisaccharides from the non-reducing ends of triple-helical β-1,3-glucans. The founder member of the family, GLU1 from L. edodes (subgroup IV) is an endo-β-1,3-glucanase with an atypical mode of substrate recognition as in the subgroup VI. Intriguingly, some fungal members from this family, such as those from Trichoderma gamsii and C. neoformans, are devoid of catalytic activity but conserve the capacity to bind short β-1,3-glucooligosaccharides (subgroup VII) [2].

Clustering of GH128

Figure 1. Clustering of the GH128 family into seven subgroups. Adapted from [2].

GH128 was created based on the study of ^^^Yuichi Sakamoto^^^ and colleagues [1]. Years later, a group headed by ^^^Mario Murakami^^^ explored the functional and structural diversity of this family [2]. For this purpose, they employed phylogenetic and Sequence Similarity Network (SSN, [3]) analyses to segregate the family into putative isofunctional subgroups. The SSN analysis resulted in two discrete clusters (subgroups VI and VII) and a third cluster that was further subdivided into five subgroups (I to V) based on SSN alignment scores and evolutionary closeness (Fig. 1). At least one member of each subgroup was biochemically and structurally characterized. Subgroups I and II were found to be predominantly present in bacteria, and the subgroups III to VII are mostly found in fungi. Bacterial enzymes are faster, feature a substrate-interacting "hydrophobic knuckle" (see #Three-dimensional structures) and attack the β-1,3-glucan in an endo mode of action, which is compatible with their biological functions (nutrition and competition). Fungal β-1,3-glucanases are known to act on remodeling of their own cell walls. Therefore, these enzymes are slower, more diverse in terms of strategies for substrate recognition (flattening mechanism – subgroups IV and VI; and hydrophobic knuckle – subgroups III, V and VII) and modes of action (exo-enzymes – subgroups III, V and VI; endo-enzymes – subgroup IV; and oligosaccharide binding proteins – subgroup VII).

Kinetics and Mechanism

As indicated by the first study of a GH128 enzyme [1], this family is part of Clan GH-A, thus suggesting that its members operate by a classical Koshland retaining mechanism. This prediction was confirmed through ¹H-nuclear magnetic resonance spectroscopy of enzymatic products [2].

Catalytic Residues

From the sequence alignment of GH128 members, two glutamic acids, E103 and E195 in L. edodes GLU1, were predicted to be the catalytic residues [1]. They were further confirmed to be the general acid/base and the catalytic nucleophile, respectively, by site-directed mutagenesis of the bacterial GH128 member from A. mediterranei [2]. These residues are located at the C-terminal ends of the strands β7 and β4 [2], as observed for other clan GH-A families.

Three-dimensional structures

A three-dimensional homology model of L. edodes GLU1 indicated similarity with several (β/α)₈-barrel (TIM-barrel) structures, including a GH39 β-xylosidase and a GH5 β-mannanase [1]. The fold resembling an (β/α)₈-barrel was further confirmed with the crystal structure determination of 9 members of the family [2]. However, in all structures, the helix α2 and the strand β3 are strictly absent [2]. Moreover, some enzymes such as the endo-β-1,3-glucanase from L. edodes (GLU1) and the exo-β-1,3-glucanase from C. neoformans, also lack the helices α1 and α3, respectively [2].

Two distinct modes of substrate binding were observed in the GH128 family [2]. The most widespread mode, termed as "hydrophobic knuckle", involves a tryptophan residue that interacts with four glucoside moieties from –5 to –2 and is fully complementary to the typically curved conformation of β-1,3-glucan chains. The other mode, only observed in fungal members belonging to subgroups IV and VI, requires substrate conformational changes to allow the binding to the catalytic interface. In these fungal subgroups, the hydrophobic knuckle is absent and two aromatic residues, positioned at the -5 and -4 subsites, create a linearized cleft, which requires a 180° torsion in the glycosidic bond between the glycosyl moieties –2 and –3 in the β-1,3-glucan chain for binding. This mode of substrate recognition is referred to as the "flattening" mechanism, due to the unusual, but also stereochemically favorable, conformation adopted by the substrate. It is notable that this mode of substrate binding has not been observed in other CAZy families active on β-1,3-glucans.

Family Firsts

First stereochemistry determination

predicted to be retaining by membership in Clan GH-A [1] and further validated by ¹H-NMR analysis of laminarin products generated by the A. mediterranei endo-β-1,3-glucanase (AmGH128_I) [2].

First catalytic nucleophile identification

predicted by sequence alignment [1] and confirmed by site-directed mutagenesis of A. mediterranei endo-β-1,3-glucanase (AmGH128_I) [2].

First general acid/base residue identification

predicted by sequence alignment [1] and confirmed by site-directed mutagenesis of A. mediterranei endo-β-1,3-glucanase (AmGH128_I) [2].

First 3-D structure

predicted by modelling of L. edodes GLU1 [1] and experimentally determined for several GH128 members including endo-β-1,3-glucanases from A. mediterranei (AmGH128_I), P. viridiflava (PvGH128_II), Sorangium cellulosum (ScGH128_II) and L. edodes (LeGH128_IV); exo-β-1,3-glucanases from B. gilchristii (BgGH128_III), C. neoformans (CnGH128_V) and A. namibiae (AnGH128_VI); and β-1,3-glucooligosaccharide binding proteins from T. gamsii (TgGH128_VII) and C. neoformans (CnGH128_VII) [2].

References

1. Sakamoto Y, Nakade K, and Konno N. (2011). Endo-β-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family. Appl Environ Microbiol. 2011;77(23):8350-4. DOI:10.1128/AEM.05581-11 |
2. Santos CR, Costa PACR, Vieira PS, Gonzalez SET, Correa TLR, Lima EA, Mandelli F, Pirolla RAS, Domingues MN, Cabral L, Martins MP, Cordeiro RL, Junior AT, Souza BP, Prates ÉT, Gozzo FC, Persinoti GF, Skaf MS, and Murakami MT. (2020). Structural insights into β-1,3-glucan cleavage by a glycoside hydrolase family. Nat Chem Biol. 2020;16(8):920-929. DOI:10.1038/s41589-020-0554-5 |
3. Atkinson HJ, Morris JH, Ferrin TE, and Babbitt PC. (2009). Using sequence similarity networks for visualization of relationships across diverse protein superfamilies. PLoS One. 2009;4(2):e4345. DOI:10.1371/journal.pone.0004345 |