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Glycoside Hydrolase Family 124

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Glycoside Hydrolase Family GH124
Clan None
Mechanism Inverting
Active site residues Catalytic acid known
CAZy DB link
http://www.cazy.org/GH124.html


Substrate specificities

Family GH124 consists of a small number of celluloytic glycoside hydrolases. The Clostridium thermocellum enzyme CtCel124A is the only member of this family that has been characterized. The enzyme is an endo-β1,4-glucanase with modest activity in vitro, but acts in synergy with the major exo-cellulase from C. thermocellum and, as a discrete entity, is able to deconstruct tobacco cell walls [1].

Kinetics and Mechanism

Using cellopentaose as the substrate and HPLC the enzyme was shown to utilize a single displacement, inverting mechanism [1].

Catalytic Residues

The catalytic general acid in CtCel124A was shown to be Glu96 based on the crystal structural of the enzyme and the observation that the E96A mutation completely inactivates the cellulase [1]. The enzyme contains no candidate catalytic general base and it was suggested that the nucleophilic water was activated by a "Grotthus-like" mechanism, as previously proposed for some GH6 cellulases [2].

Three-dimensional structures

The enzyme displays a superhelical fold in which a constellation of α-helices encircling a central helix that houses the catalytic apparatus. This fold was described as having closest similarity to black swan lysozyme G (Lyz23) from family GH23 [1]; similarities also exist to family GH19 chitinases, family GH22 lysozymes, and family GH134 β-mannanases. The catalytic acid, Glu96, is located at the C-terminus of the central helix. The substrate-binding cleft can be divided into two discrete topographical domains in which the bound cellotriose molecules display twisted and linear conformations, respectively, suggesting that the enzyme may target the interface between crystalline and disordered regions of cellulose. The active site of CtCel124 displays remarkable structural conservation with a GH23 lytic transglycosylase [1].

Family Firsts

First stereochemistry determination
The sterochemical outcome of catalysis is inversion of the β linkage into the α anomer [1]. The method used to detect the stereochemical outcome was the HPLC method of Braun et al. [3]. The reaction was too slow to detect through NMR as rapid mutarotation obscured the sterochemistry of glycosidic bond cleavage.
First general base residue identification
The candidate catalytic base has not been identified.
First general acid/base residue identification
The catalytic general acid was identified from analysis of structure in harness with mutagenesis data and sequence analysis [1].
First 3-D structure
The X-ray crystal structure of CtCel124 was determined in complex with two cellotriose molecules. The structure revealed a novel cellulase fold and hinted that the enzyme attacks cellulose at the interface between two structural domains of the substrate [1].

References

  1. Brás JL, Cartmell A, Carvalho AL, Verzé G, Bayer EA, Vazana Y, Correia MA, Prates JA, Ratnaparkhe S, Boraston AB, Romão MJ, Fontes CM, and Gilbert HJ. Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis. Proc Natl Acad Sci U S A. 2011 Mar 29;108(13):5237-42. DOI:10.1073/pnas.1015006108 | PubMed ID:21393568 | HubMed [Bras2011]
  2. Koivula A, Ruohonen L, Wohlfahrt G, Reinikainen T, Teeri TT, Piens K, Claeyssens M, Weber M, Vasella A, Becker D, Sinnott ML, Zou JY, Kleywegt GJ, Szardenings M, Ståhlberg J, and Jones TA. The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175. J Am Chem Soc. 2002 Aug 28;124(34):10015-24. PubMed ID:12188666 | HubMed [Koivula2002]
  3. Braun C, Meinke A, Ziser L, and Withers SG. Simultaneous high-performance liquid chromatographic determination of both the cleavage pattern and the stereochemical outcome of the hydrolysis reactions catalyzed by various glycosidases. Anal Biochem. 1993 Jul;212(1):259-62. DOI:10.1006/abio.1993.1320 | PubMed ID:8368500 | HubMed [Braun1993]
All Medline abstracts: PubMed | HubMed
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