Glycoside Hydrolase Family 76

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• Author: ^^^Spencer Williams^^^
• Responsible Curators: ^^^Gideon Davies^^^ and ^^^Harry Gilbert^^^

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Glycoside Hydrolase Family GH76
Clan	not assigned
Mechanism	retaining
Active site residues	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 arising 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. Crystallographic evidence from a binary complexes of B. circulans GH76 with substrate and inhibitors and complemented by calculations of preferred conformations on enzyme supports a ¹S₅→B_2,5^‡→^OS₂ conformational reaction coordinate [6].

Catalytic Residues

A pair of conserved aspartic acid residues (D124 and D125 in the catalytic domain of Bacillus circulans GH76) have been assigned as nucleophile and acid/base, respectively, based on X-ray analysis of substrate and inhibitor complexes, dovetailed with kinetic analysis of mutants [6].

Three-dimensional structures

Three-dimensional structures are available for several bacterial members of GH76, including Bacillus circulans, Bacteroides thetaiotaomicron and Listeria innocua Clip11262 (see the GH76 structure page in the CAZy Database). They have an (α/α)₆ fold. A complex of mannopentaose bound in the active site of Bacillus circulans GH76 defined the -4 to +1 subsites, and showed the sugar binding in the -1 subsite in a ¹S₅ conformation and a complex with α-mannosyl-1,6-isofagomine with the isofagomine ring in a B_2,5 conformation [6].

Family Firsts

First stereochemistry determination

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

First catalytic nucleophile identification

D124 for Bacillus circulans catalytic domain [6].

First general acid/base residue identification

D125 for Bacillus circulans catalytic domain [6].

First 3-D structure of a GH76 enzyme

Listeria innocua Lin0763 (unpublished, PDB ID 3k7x)

References

1. Maruyama Y and Nakajima T. (2000). The aman6 gene encoding a yeast mannan backbone degrading 1,6-alpha-D-mannanase in Bacillus circulans: cloning, sequence analysis, and expression. Biosci Biotechnol Biochem. 2000;64(9):2018-20. DOI:10.1271/bbb.64.2018 |
2. Nakajima T, Maitra SK, and Ballou CE. (1976). An endo-alpha1 leads to 6-D-mannanase from a soil bacterium. Purification, properties, and mode of action. J Biol Chem. 1976;251(1):174-81. | Google Books | Open Library
3. Yokoyama K and Ballou CE. (1989). Synthesis of alpha 1----6-mannooligosaccharides in Mycobacterium smegmatis. Function of beta-mannosylphosphoryldecaprenol as the mannosyl donor. J Biol Chem. 1989;264(36):21621-8. | Google Books | Open Library
4. Cuskin F, Lowe EC, Temple MJ, Zhu Y, Cameron E, Pudlo NA, Porter NT, Urs K, Thompson AJ, Cartmell A, Rogowski A, Hamilton BS, Chen R, Tolbert TJ, Piens K, Bracke D, Vervecken W, Hakki Z, Speciale G, Munōz-Munōz JL, Day A, Peña MJ, McLean R, Suits MD, Boraston AB, Atherly T, Ziemer CJ, Williams SJ, Davies GJ, Abbott DW, Martens EC, and Gilbert HJ. (2015). Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism. Nature. 2015;517(7533):165-169. DOI:10.1038/nature13995 |
5. Kitagaki H, Wu H, Shimoi H, and Ito K. (2002). Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae. Mol Microbiol. 2002;46(4):1011-22. DOI:10.1046/j.1365-2958.2002.03244.x |
6. Thompson AJ, Speciale G, Iglesias-Fernández J, Hakki Z, Belz T, Cartmell A, Spears RJ, Chandler E, Temple MJ, Stepper J, Gilbert HJ, Rovira C, Williams SJ, and Davies GJ. (2015). Evidence for a boat conformation at the transition state of GH76 α-1,6-mannanases--key enzymes in bacterial and fungal mannoprotein metabolism. Angew Chem Int Ed Engl. 2015;54(18):5378-82. DOI:10.1002/anie.201410502 |