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Glycoside Hydrolase Family 44

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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

Substrate specificities

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. [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]. GH44 enzymes act with retention of anomeric stereochemistry [1], 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.

Catalytic Residues

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 [1], 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 [2].
  • 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 [5].

Three-dimensional structures

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 [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].

GH44 was previously known as cellulase family J; see [6] or the ExPASy page on GH families.

Family Firsts

First stereochemistry determination
Kitago et al. [1] 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. [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.


  1. 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 | PubMed ID:17905739 [Kitago2007]
  2. 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 | PubMed ID:19345197 [Nam2009]
  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 | PubMed ID:16314409 [Najmudin2006]
  4. 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 | PubMed ID:22112767 [Warner2011]
  5. 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 | PubMed ID:19915043 [Warner2010]
  6. 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 | PubMed ID:8352747 [Henrissat1993]

All Medline abstracts: PubMed