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Glycoside Hydrolase Family 76

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Glycoside Hydrolase Family GH76
Clan not assigned
Mechanism retaining
Active site residues known
CAZy DB link

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 a Bacteroides thetaiotaomicron α-mannanase [4]. GH76 enzymes are believed to proceed through a classical Koshland double-displacement mechanism. Crystallographic evidence from a binary complexes of the catalytic domain of Bacillus circulans Aman6 with substrate and inhibitors, complemented by quantum mechanics/molecular mechanics calculations of preferred conformations on-enzyme supports a 1S5B2,5OS2 conformational reaction coordinate [6].

Catalytic Residues

Inspection of the X-ray structure of Bacteroides thetaiotaomicron BT3792 revealed two consecutive asparate residues, D258 and D259, that were predicted to be catalytic residues [4]. The equivalent pair of conserved aspartic acid residues (D124 and D125 in the catalytic domain of Bacillus circulans Aman6) were identified as catalytic nucleophile and general 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 the catalytic domain of Bacillus circulans Aman6, Bacteroides thetaiotaomicron BT2949 and BT3792, 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. A complex of the same enzyme with α-1,6-mannobiose showed the disaccharide binding in the -3/-2 subsites (unpublished, PDB ID 4boj). Several inhibtior complezes have been reported. A complex with the inhibitor α-mannosyl-1,6-isofagomine revealed the inhibtior to bind in the -2/-1 subsites and displayed the isofagomine ring in a B2,5 conformation, with the nitrogen of the inhibitor hydrogen-bonded to the nucleophile (D124) [6]. The S-linked analogue α-mannosyl-1,6-S-isofagomine (ManSIFG) bound with similar affinity to the enzyme and also in the -2/-1 subsites, but instead the inhibitor bound in a relaxed 4C1 conformation with the inhibitor nitrogen hydrogen bonded to the acid/base (D125) [7].

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)


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  1. Error fetching PMID 11055417: [Maruyama2000]
  2. Error fetching PMID 811665: [Nakajima1976]
  3. Error fetching PMID 2480954: [Yokoyama1989]
  4. Error fetching PMID 25567280: [Cuskin2015]
  5. Error fetching PMID 12421307: [Kitagaki2002]
  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. 54, 5378-82. DOI:10.1002/anie.201410502 | PubMed ID:25772148 | HubMed [Thompson2015]
  7. Error fetching PMID 28766587: [Belz2017]
All Medline abstracts: PubMed | HubMed