Difference between revisions of "Glycoside Hydrolase Family 12"

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Authors: ^^^Gerlind Sulzenbacher^^^ and ^^^Mats Sandgren^^^
Responsible Curator: ^^^Gerlind Sulzenbacher^^^

Glycoside Hydrolase Family GH12
Clan	GH-C
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH12.html

Substrate specificities

The substrate specificities found among the glycoside hydrolases of family 12 are: endo-β-1,4-glucanase (EC 3.2.1.4), xyloglucan endo-hydrolase (EC 3.2.1.151), endo-β-1,3-1,4-glucanase (EC 3.2.1.73). Xyloglucan endo-transglycosylase (XET, EC 2.4.1.207) activity has been observed in a single fungal GH12 member (GenBank AAN89225.1) using a XET-specific screen [1], although this may represent a side activity of a predominant xyloglucan endo-hydrolase [2, 3].

Kinetics and Mechanism

GH12 enzymes are retaining enzymes, as first shown by NMR studies [4] on endoglucanase 3 from Humicola insolens, and is believed to follow a classical Koshland double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and subsequently this intermediate is hydrolysed via oxocarbenium-ion transition states. No detailed studies involving both steady state and pre-steady state kinetic have yet been reported for GH12.

Catalytic Residues

The catalytic nucleophile and the general acid/base catalyst of GH12 enzymes was initialy predicted by sequence homology to the xylanase members of GH11, a glycoside hydrolase family where the catalytic nucleophile was first identified in the Bacillus circulans endo-xylanase through trapping of the 2-deoxy-2-fluoroxylobiosyl-enzyme intermediate and subsequent peptide mapping via LC-MS/MS technologies [5]. GH11 and GH12 together form clan GH-C. The prediction of the catalytic nucleophile and the general acid/base of GH family 12 enzymes was later confirmed to be correct when the first three dimensional structure of a GH family 12 enzyme was determined, that of Streptomyces lividans CelB2 [6]. The catalytic nucleophile was subsequentialy confirmed in Streptomyces lividans CelB2 to be Glu 120 by using the same labeling strategy used for detecting the catalytic nucleophile of family GH11 [7].

Three-dimensional structures

Figure 1: Streptomyces lividans CelB2 in complex with a 2-deoxy-2-fluorocellotrioside (PDB 2NLR). Two distinct species were observed in the crystal: the glycosyl-enzyme intermediate with the mechanism-based inhibitor covalently linked to the nucleophile Glu 120, and the reaction product 2-deoxy-2-fluoro-cellotriose. Colour coding: CelB2 in rainbow colour-ramping from the N-terminus in blue to the C-terminus in red; catalytic residues in magenta; bound oligosaccharide in black

Enzymes from family GH12 adopt a “jelly-roll” fold with two twisted, mostly antiparallel β-sheets stacking on top of each other and enclosing a long substrate-binding groove located on the concave face of the sheet (Fig. 1). A single α-helix packs against the convex surface of the outer β-sheet. GH12 members of bacterial origin feature an additional two-stranded β-sheet, stabilized by a disulphide bridge, located at the rim of the substrate binding groove and providing an additional substrate binding platform as compared to the eukaryotic counterparts.

The first structural template provided for family GH12 was that of Streptomyces lividans CelB2 in the apo-form [6] and in the form of a glycosyl-enzyme intermediate [8]. This first structure confirmed previous predictions, based on hydrophobic cluster analysis [9], that GH12 enzymes would display significant structural similarity with family GH11 xylanases and gave rise to the creation of clan GH-C, which so far comprises only the two above families. The first structure of a fungal GH12 enzyme was provided shortly after for Trichoderma reesei (formerly Hypocrea jecorina) Cel12A [10]. To date structural models, both in the apo-form and in complex with oligosaccharides, are available for sixteen family members.

From the different complex structures it can be seen that the substrate-binding groove of family GH12 enzymes harbours six binding sites, from -4 to +2, lined by numerous aromatic residues providing staking platforms for the individual pyranose units. This is consistent with the endoglucanase activity of these enzymes, exhibiting low catalytic efficiency towards short chain substrates [11]. The catalytic nucleophile Glu120 (S. lividans CelB2 numbering) and the general acid/base Glu203 are in close proximity of an invariant aspartate, which might have an important role in catalysis and the constellation of this catalytic triad is reminiscent of the catalytic centre of family GH7 enzymes. Like in family GH7 enzymes the acid/base residues of family GH12 members protonate syn to the pyranoside 0-5-C-1 bond.

Figure 2: Xyloglucanase XG12 from Bacillus licheniformis in complex with xyloglucan (PDB 2JEN). α-1,6-xylose substitutions are present at subsites -3, -2, +1 and +2.

For xyloglucan specific enzymes of the family, structural and functional studies of xyloglucanase XG12 from Bacillus licheniformis [12] and the xyloglucan specific endo-β-1,4-glucanase XEG1 from Aspergillus aculeatus KSM 50 [13] have shown that α-1,6-xylose substitutions can be tolerated at subsites -3, -2, +1 and +2 (Fig. 2). Certain GH12 enzymes exhibit β-1,3-1,4-glucanase activity and the crystal structure of Humicola grisea Cel12A in complex with a mixed linkage oligosaccharide arising from a transglycosylation reaction revealed that the β-1,3-linkage can be accommodated with ease between subsites -3 and -2 [14]. Speculations had been forwarded that a long loop crossing the substrate-binding groove at its reducing end, known as the “cord” and conserved in all clan GH-C members, might undergo conformational changes upon substrate binding [15], but the available GH12 complex structures support the view that the role of this loop is to deliver residues for substrate binding.

The crystal structure of Aspergillus aculeatus XEG1 in complex with the glycoside hydrolase inhibitory protein from carrot. EDGP, reveals an original mode of inhibition, distinct from the one observed previously for the inhibition of a family GH11 xylanase by a wheat protein [16]. In the XEG1-GHIP complex the inhibitor mimics a xyloglucan substrate and inserts two conserved arginine residues into the active site, which establish salt bridges with the carboxylate groups of the catalytic center (Fig. 3) [13]).

Figure 3: Inhibition of Aspergillus aculeatus XEG1 by the glycoside hydrolase inhibitory protein from carrot. EDGP (PDB 3VLB). Two conserved arginine residues of the inhibitor make salt bridges with the catalytic residues of XEG1. Colour coding: XEG1 in pink with carboxylate groups of the catalytic centre in purple; EDGP in lightblue with the conserved arginine residues in deepblue.

Due to their commercial interest in bioprocessing applications, GH12 endo-β-1,4-glucanases from Humicola grisea [17], Trichoderma reesei [17, 18], Rhodothermus marinus [19], Pyrococcus furiosus [20] and Thermotoga maritima [21] have been extensively engineered and the structural studies of the resulting variants have provided valuable insight into structural features governing enzyme activity and stability.

Family Firsts

First sterochemistry determination: Humicola insolens endoglucanase 3 by NMR [4].

First catalytic nucleophile identification: The catalytic nucleophile was first identified in Streptomyces lividans CelB2 by trapping of a glycosyl-enzyme intermediate followed by X-ray structure determination [8] and peptide mapping via LC-MS/MS technologies [7].

First general acid/base residue identification: inferred from homology with family GH11 enzymes

First 3-D structure: The crystal structure of Streptomyces lividans CelB2 [6]

References

All Medline abstracts: PubMed

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 * [[Responsible Curator]]:  ^^^Gerlind Sulzenbacher^^^
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