Glycoside Hydrolase Family 44

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• Author: ^^^Peter Reilly^^^
• Responsible Curator: ^^^Peter Reilly^^^

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Glycoside Hydrolase Family GH44
Clan	None specified, but Kitago et al. [1] and Nam et al. [2] suggest that it belongs to Clan GH-A.
Mechanism	Retaining
Active site residues	Catalytic proton donor/acceptor: Glu Catalytic nucleophile: Glu
CAZy DB link
https://www.cazy.org/GH44.html

Substrate specificities

Active on many substances, including cellooligosaccharides of DP4 and longer, carboxymethylcellulose, xylan, lichenan, Avicel (slightly), and xyloglucan, which appears to be a prime substrate [3, 4].

Kinetics and Mechanism

The most complete analyses of kinetics on various substrates are by Najmudin et al. [3] and by Warner et al. [4, 5]. GH44 endoglucanases are also xyloglucanases. They hydrolyze longer cellooligosaccharides faster than shorter cellooligosaccharides [4, 5]. They act asymmetrically on cellooligosaccharides, for instance producing more cellobiose and cellotetraose than cellotriose from cellohexaose [4, 5], with substrates bound with more of their residues in negatively-numbered than in positively-numbered subsites. Furthermore, disproportionation occurs, with more cellotetraose than cellobiose formed from cellohexaose, evidently caused by formation of larger unobserved products that are then rapidly hydrolyzed [4, 5]. The mechanism is retaining [1], with a covalent bond being formed between the catalytic nucleophile and the C1' atom, leading to liberation of the leaving group; subsequently, the glycosyl-enzyme is cleaved by water.

Catalytic Residues

The catalytic residues in this family have been suggested by several experiments with diverse enzymes. These include:

• 'Clostridium thermocellum endoglucanase: Catalytic proton donor/acceptor, Glu186; catalytic nucleophile, Glu359; by soaking the wild-type crystals with cellopentaose or cellohexaose and noting the positions of the residues relative to the reducing end of the cellotetraose product [1], and also by finding no activity with E186Q and E359Q mutants. * Protein from metagenomic library: Catalytic proton donor/acceptor, Glu221; catalytic nucleophile, Glu393 by location in the active site of the wild-type crystal structure [2].
• 'Clostridium acetobutylicum xyloglucanase/endoglucanase: Catalytic proton donor/acceptor, Glu180; catalytic nucleophile, Glu352 also by location in the crystal structure of the wild-type enzyme, and by comparison with the C. thermocellum structure [5].

Three-dimensional structures

The first three-dimensional structure was by Kitago et al., who found a TIM-like barrel domain and a beta-sandwich domain in C. thermocellum endoglucanase [1]. Similar structures were found by Nam et al. [2] in a protein from a metagenomic library and by Warner et al. [5] in C. acetobutylicum endoglucanase. Ca⁺⁺ and Zn⁺⁺ ions are found as ligands [1].

Family Firsts

First stereochemistry determination

Kitago et al. [1] found that C. thermocellum endoglucanase acts by a retaining mechanism. They observed that a beta-anomer was preferentially formed during cyclohexaitol hydrolysis.

First catalytic nucleophile identification

Kitago et al. [1], by testing activity of the C. thermocellum endoglucanase E359Q mutant.

First general acid/base residue identification

Kitago et al. [1], by testing activity of the C. thermocellum endoglucanase E186Q mutant.

First 3-D structure

Kitago et al. [1] of C. thermocellum endoglucanase. It had a resolution of 0.96 Å and allowed the identification of the catalytic residues and the mechanism.

References

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3. Najmudin S, Guerreiro CI, Carvalho AL, Prates JA, Correia MA, Alves VD, Ferreira LM, Romão MJ, Gilbert HJ, Bolam DN, and Fontes CM. (2006). Xyloglucan is recognized by carbohydrate-binding modules that interact with beta-glucan chains. J Biol Chem. 2006;281(13):8815-28. DOI:10.1074/jbc.M510559200 |

4. Warner CD, Go RM, García-Salinas C, Ford C, and Reilly PJ. Kinetic characterization of a glycoside hydrolase family 44 xyloglucanase/endoglucanase from Ruminococcus flavefaciens FD-1. Enzyme Microb Technol 2011 Jan 5; 48 (1) 27-32. doi:10.1016/j.enzmictec.2010.08.009 pmid: .