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Glycoside Hydrolase Family 134

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Glycoside Hydrolase Family GH134
Clan none
Mechanism inverting
Active site residues known
CAZy DB link

Substrate specificities

Glycoside hydrolase family 134 exclusively contains β-1,4-mannanases, and was created based on the discovery of Man134A from Aspergillus nidulans [1]. Man134A exhibits weak activity on galactomannan but robust activity on glucomannan, and activity on β-1,4-linked mannopentaose and hexaose [1]. SsGH134 from Streptomyces sp. NRRL B-24484 possesses a strong preference for unsubstituted linear β-mannans over gluco- and galactomannans [2]. SsGH134 possesses activity on β-1,4-linked mannotetraose, pentaose and hexaose.

Kinetics and Mechanism

1H NMR spectroscopic analysis of SsGH134 catalyzed cleavage of a benzoylhydrazine derivative of β-mannohexaose provided evidence for the formation of the α-anomer, consistent with an inverting mechanism [2]. Crystallographic evidence from binary complexes of SsGH134 with substrate and product, complemented by quantum mechanics/molecular mechanics calculations, supports a "southern hemisphere" 1C43H43S1 conformational itinerary along the reaction coordinate [2].

Catalytic Residues

Structural analysis of SsGH134 from Streptomyces sp. (see below) suggested roles for Glu45 as a general acid catalyst and for Asp57 as a general base catalyst [2]. These roles were supported by mutagenesis with the corresponding Glu45Ala and Asp57Ala single mutants inactive on β-mannopentaose and hexaose.

Three-dimensional structures

The three dimensional structure was first reported for SsGH134, and exists as a mixed α-helix/β-sheet fold with resemblance to HEWL (GH22), as well as GH19 chitinases, GH23 G-type lysozyme, and GH124 cellulases [2]. A binary Michaelis complex of the E45A variant of SsGH134 with mannopentaose bound in the active site defined the -3 to +2 subsites, and showed the sugar binding in the -1 subsite in a 1C4 conformation; a similar conformation was observed for the reducing-end mannose of a complex of mannotriose bound to the -3 to -1 subsites of wild-type SsGH134 [2]. Within the Michaelis complex, the carboxylate of the highly conserved C-terminal residue Ile173 is hydrogen bonded to O6 of the –2 mannosyl unit, and also forms a salt-bridge with the highly conserved K59. Within only the Michaelis complex, this interaction results in the formation of an active cleft tunnel reminiscent of processive glycoside hydrolases; the C-terminal segment is disordered in both apo and mannotriose product-bound forms [2]. SsGH134 can be classified as a syn protonator, with the acid residue situated syn to the C1-O5 bond.

Family Firsts

First stereochemistry determination
SsGH134 was shown to be inverting by 1H NMR spectroscopy [2].
First general acid residue identification
Glu45 in SsGH134 by structural study supported by kinetic analysis of mutants [2].
First general base residue identification
Asp57 in SsGH134 by structural study supported by kinetic analysis of mutants [2].
First 3-D structure
SsGH134 from Streptomyces sp. (PDB ID 5JTS) [2].


  1. Shimizu M, Kaneko Y, Ishihara S, Mochizuki M, Sakai K, Yamada M, Murata S, Itoh E, Yamamoto T, Sugimura Y, Hirano T, Takaya N, Kobayashi T, and Kato M. (2015). Novel β-1,4-Mannanase Belonging to a New Glycoside Hydrolase Family in Aspergillus nidulans. J Biol Chem. 2015;290(46):27914-27. DOI:10.1074/jbc.M115.661645 | PubMed ID:26385921 [Shimizu2015]
  2. Jin Y, Petricevic M, John A, Raich L, Jenkins H, Portela De Souza L, Cuskin F, Gilbert HJ, Rovira C, Goddard-Borger ED, Williams SJ, and Davies GJ. (2016). A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism. ACS Cent Sci. 2016;2(12):896-903. DOI:10.1021/acscentsci.6b00232 | PubMed ID:28058278 [Jin2016]

All Medline abstracts: PubMed