Glycoside Hydrolase Family 76

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• Author: ^^^Spencer Williams^^^
• Responsible Curator: ^^^Gideon Davies^^^ ^^^Harry Gilbert^^^

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Glycoside Hydrolase Family GH76
Clan	not assigned
Mechanism	retaining
Active site residues	not known
CAZy DB link
https://www.cazy.org/GH76.html

Substrate specificities

Glycoside hydrolases of family GH76 are endo-acting α-mannanases. GH76 genes are found within bacteria and fungi.

Bacterial GH76 enzymes cleave α-1,6-mannans, such as those found within the α-1,6-linked backbone of fungal mannoproteins and mycobacterial cell wall lipomannan and lipoarabinomannan.

This family was originally created from the cloning and characterization of Aman6 from Bacillus circulans TN-31 [1], which appears to be the same enzyme as that characterized much earlier by Ballou and co-workers [2]. Aman6 degrades α-1,6-mannan to a mixture of the mannobiose and mannotriose [1]; mannotriose is the minimum substrate for the enzyme [2]. A possible GH76 enzyme has been detected within Mycobacterium smegmatis, which has the capacity to degrade α-1,6-mannooligosaccharides [3].

Additional characterized GH76 enzymes include several from Bacteroides thetaiotaomicron [4]. B. thetaiotaomicron expresses numerous GH76 enzymes. Several of these are found within polysaccharide utilization loci that are specifically up-regulated upon exposure to yeast α-mannan. These enzymes have the capacity to utilize unadorned linear α-1,6-mannan, but have little activity on highly branched wildtype α-mannan. Certain B. thetaiotaomicron GH76 enzymes are lipoenzymes that are associated with the cell surface, where they appear to act on large yeast mannan molecules that have undergone partial trimming to expose sections of the core α-1,6-mannan. Other periplasmic located GH76 enzymes have activity on shorter α-1,6-mannan fragments, which are obtained by importation of partially-digested fragments from the action of cell surface enzymes.

Fungal GH76 enzymes have been speculated to be involved in cross-linking of GPI-anchored proteins into the cell wall, where they are proposed to act as transglycosylases [5]. No biochemical data has been obtained for any fungal GH76 enzyme in support of the so-called anchorage hypothesis.

Kinetics and Mechanism

Family GH76 endo- α-mannosidases are retaining enzymes, as first shown by ¹H NMR analysis of the hydrolysis of 4-nitrophenyl α-mannosyl-1,6-α-mannopyranoside by Bacteroides thetaiotaomicron [4]. GH76 enzymes are believed to proceed through a classical Koshland double-displacement mechanism.

Catalytic Residues

A pair of conserved aspartic acid residues (D258 and D259) have been proposed as acid/base and nucleophile based on analysis of the X-ray structures of GH76 members [4].

Three-dimensional structures

Three-dimensional structures are available for several bacterial members of GH76, including Bacillus circulans, Bacteroides thetaiotaomicron and Listeria innocua" Clip11262 [6]. They have an (α/ α)₆ fold. A ligand bound structure with &alpha-1,6-mannobiose is available for the B. circulans enzyme. This structure appears to involve binding within the active site cleft although it does not appear to involve ligand binding at the catalytic site.

Family Firsts

First stereochemistry determination

Bacteroides thetaiotaomicron α-1,6-mannanase by ¹H NMR [4]

First catalytic nucleophile identification

no experimental evidence

First general acid/base residue identification

no experimental evidence

First 3-D structure of a GH76 enzyme

Listeria innocua Lin0763 [6]

References

1. pmid= 11055417

2. pmid= 811665

3. pmid= 2480954

4. Cuskin, F., Lowe, E.C., Temple, M.J., Zhu, Y., Pudlo, N.A., Cameron, E.A., Porter, N.T., Urs, K., Thompson, A.J., Cartmell, A., Rogowski, A., Tolbert, T., Piens, K., Bracke, D., Vervecken, W., Hakki, Z., Speciale, G., Munōz-Munōz, J.L, Peña, M.J., McLean, R., Suits, M.D., Boraston, A.B., Atherly, T., Ziemer, C.J., Williams, S.J., Davies, G.J., Abbott, D.W., Martens, E.C., Gilbert, H.J., Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism, Nature, 2015, 517, 165. URL http://dx.doi.org/10.1038/nature13995

5. pmid= 12421307

6. PDB link for 3K7X; URL http://www.rcsb.org/pdb/explore/explore.do?structureId=3K7X