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Glycoside Hydrolase Family 44
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|Glycoside Hydrolase Family GH44|
|Clan||None specified, but |
Kitago et al.  and Nam et al. 
suggest that it belongs to Clan GH-A.
|Active site residues||Catalytic proton donor/acceptor: Glu|
Catalytic nucleophile: Glu
|CAZy DB link|
GH44 glycoside hydrolases are active on many substances, including tetrasaccharide cellooligosaccharides and longer oligomers, carboxymethylcellulose, xylan, lichenan, Avicel (slightly), and xyloglucan, the last of which appears to be a prime substrate [3, 4].
Kinetics and Mechanism
The most complete analyses of GH44 kinetics on various substrates are by Najmudin et al.  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]. GH44 enzymes act with retention of anomeric stereochemistry , through a classical Koshland double-displacement mechanism with a covalent bond being formed between the catalytic nucleophile and the anomeric carbon of the substrate, leading to liberation of the leaving group; subsequently, the glycosyl-enzyme is cleaved by water. A general acid/base residue acts as a general acid in the first step to assist departure of the aglycon; in the second step this residue acts as a general base to assist in deprotonating a nucleophilic water residue.
The catalytic residues in this family have been suggested by several experiments with diverse enzymes. These include:
- Clostridium thermocellum endoglucanase: General acid/base, 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 , and also by finding no activity with E186Q and E359Q mutants.
- Protein from metagenomic library: General acid/base, Glu221; catalytic nucleophile, Glu393 by location in the active site of the wild-type crystal structure .
- Clostridium acetobutylicum xyloglucanase/endoglucanase: General acid/base, Glu180; catalytic nucleophile, Glu352 also by location in the crystal structure of the wild-type enzyme, and by comparison with the C. thermocellum structure .
The first three-dimensional structure was by Kitago et al., who found a TIM-like barrel domain and a β-sandwich domain in C. thermocellum endoglucanase . Similar structures were found by Nam et al.  in a protein from a metagenomic library and by Warner et al.  in C. acetobutylicum endoglucanase. Ca++ and Zn++ ions are found as ligands .
- First stereochemistry determination
- Kitago et al.  found that C. thermocellum endoglucanase acts by a retaining mechanism. They observed that a β-anomer was preferentially formed during cyclohexaitol hydrolysis.
- First catalytic nucleophile identification
- Kitago et al. , by testing activity of the C. thermocellum endoglucanase E359Q mutant.
- First general acid/base residue identification
- Kitago et al. , by testing activity of the C. thermocellum endoglucanase E186Q mutant.
- First 3-D structure
- Kitago et al.  of C. thermocellum endoglucanase. It had a resolution of 0.96 Å and allowed the identification of the catalytic residues and the mechanism.
- Kitago Y, Karita S, Watanabe N, Kamiya M, Aizawa T, Sakka K, and Tanaka I. (2007). Crystal structure of Cel44A, a glycoside hydrolase family 44 endoglucanase from Clostridium thermocellum. J Biol Chem. 2007;282(49):35703-11. DOI:10.1074/jbc.M706835200 |
- Nam KH, Kim SJ, and Hwang KY. (2009). Crystal structure of CelM2, a bifunctional glucanase-xylanase protein from a metagenome library. Biochem Biophys Res Commun. 2009;383(2):183-6. DOI:10.1016/j.bbrc.2009.03.149 |
- 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 |
- Warner CD, Go RM, García-Salinas C, Ford C, and Reilly PJ. (2011). Kinetic characterization of a glycoside hydrolase family 44 xyloglucanase/endoglucanase from Ruminococcus flavefaciens FD-1. Enzyme Microb Technol. 2011;48(1):27-32. DOI:10.1016/j.enzmictec.2010.08.009 |
- Warner CD, Hoy JA, Shilling TC, Linnen MJ, Ginder ND, Ford CF, Honzatko RB, and Reilly PJ. (2010). Tertiary structure and characterization of a glycoside hydrolase family 44 endoglucanase from Clostridium acetobutylicum. Appl Environ Microbiol. 2010;76(1):338-46. DOI:10.1128/AEM.02026-09 |
- Henrissat B and Bairoch A. (1993). New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1993;293 ( Pt 3)(Pt 3):781-8. DOI:10.1042/bj2930781 |