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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. Novel β-1,4-Mannanase Belonging to a New Glycoside Hydrolase Family in Aspergillus nidulans. J Biol Chem. 2015 Nov 13;290(46):27914-27. DOI:10.1074/jbc.M115.661645 | PubMed ID:26385921 | HubMed [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. A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism. ACS Cent Sci. 2016 Dec 28;2(12):896-903. DOI:10.1021/acscentsci.6b00232 | PubMed ID:28058278 | HubMed [Jin2016]
All Medline abstracts: PubMed | HubMed
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