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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" 1C4→3H4‡→3S1conformational 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].
