Glycoside Hydrolase Family 81

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Author: ^^^Julie Grondin^^^
Responsible Curator: ^^^Al Boraston^^^

Glycoside Hydrolase Family GH81
Clan	none
Mechanism	inverting
Active site residues	not known
CAZy DB link
http://www.cazy.org/GH81.html

Substrate specificities

GH81 family are endo-β(1,3)-glucanases (EC 3.2.1.39) with diverse physiological roles, such as plant biomass degradation, cell cycling, and enzymatic pathogen defense. They are mostly found in bacteria and fungi, particularly abundant in Saccharomyces, and Streptomyces species. Activity has been demonstrated on laminarin [1, 2, 3, 4, 5, 6], curdlan [1, 3, 5, 6], and pachyman [2, 5].

Kinetics and Mechanism

GH81 enzymes follow an inverting mechanism, first shown by ¹H-NMR during the hydrolysis of laminarioligosaccharides [7], and laminarin [2], thus operating by a single-displacement mechanism.

Catalytic Residues

Primary sequence alignments of GH81 reveal a number of highly conserved residues, including two glutamate residues and one aspartate residue which are located in the active site cleft and likely to serve as catalytic residues [3, 4, 8].

Mutagenesis experiments, chemical rescue, and examination of the active site architecture and product conformational itinerary of ligand complexes strongly suggest that one of the two glutamic acids (E542 in GH81 from Bacillus halodurans C-125) acts as the catalytic base by activating a correctly positioned catalytic water, and the aspartic acid (D422 in BhGH81) acts as the catalytic acid [5, 6, 8]. While mutagenesis studies show that mutation of the second glutamic acid (E546 in BhGH81) results in a dramatic reduction in activity [8], there is conflicting evidence for the role of this residue in catalysis. Pluvinage et al. report that this residue is not correctly positioned to assist in catalysis [5], while Zhou et al. proposes that this residue is the catalytic acid, as the distance between the two glutamic acids is ideal for the inverting mechanism displayed by these enzymes [4]. As such, the exact identity of the catalytic residues for GH81 enzymes remains unclear.

Three-dimensional structures

Figure 1. Structure of Lam81A from Rhizomucor meihei (PDB ID 4K3A). Domain A (blue) and Domain C (green) comprise the core of the enzyme, with domain B (orange) acting as a stabilizer. The proposed catalytic residues are shown as red sticks.

GH81 are multimodular, although the composition of the domains can vary. The first characterized structure, Lam81A from Rhizomucor miehei CAU432, comprises an N-terminal β-sandwich domain (domain A), a small α/β domain (domain B), and a C-terminal (α/α)₆ domain (domain C) [4]. Domains A and C form the core of the enzyme (likely stabilized by domain B), each contributing residues to the active and substrate binding sites (Figure 1). This architecture is largely conserved in the GH81 from Bacillus halodurans C-125 (BhGH81) [5], and the cellulosomal GH81 from Clostridium themocellum ATCC 27405 (CtLam81A) [6], however, the C-terminal domain is a CBM56 in BhGH81 and a cellulosomal dockerin in CtLam81A.

GH81 structures are unique among GHs and also differ from other characterized endo-β(1,3)-glucanases in the PDB. As such, GH81 is not classified into any GH clan.

Figure 2. The structure of GH81 from Bacillus halodurans suggests that GH81 are capable of binding helical forms of β-glucans PDB ID 5T4G). The triple helical structure of curdlan (beige, yellow, cyan) is shown, with the pitch (16Å) and spacing (5.3Å) between strands indicated. Figure from []

Spanning domains A and C is a large cleft (10Å deep, 10Å wide, 70Å long), in which the proposed catalytic residues are located. Extensive co-crystallization of BhGH81 in complex with a range of laminarioligosaccharides clearly defines the catalytic and ancillary binding subsites, and also provides structural evidence for the ability of this enzyme for to generate a pool of oligosaccharide products [5]. Notably, these structures reveal ability of this enzyme to simultaneously bind oligosaccharides in the active and ancillary binding sites, and further suggests that this enzyme binds and cleaves helical forms of β-1,3-glucans in an endo-processive manner [5] (Figure 2).

Family Firsts

First stereochemistry determination: β-glucan binding protein (GBP) from soybean (Glycine max L.) by ¹H-NMR [7].

First 3-D structure: Lam81A from Rhizomucor miehei CAU432 [4].

References

All Medline abstracts: PubMed