Glycoside Hydrolase Family 48

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• Author: ^^^Bareket Dassa^^^
• Responsible Curator: ^^^Ed Bayer^^^

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Glycoside Hydrolase Family GH48
Clan	GH-M
Mechanism	inverting
Active site residues	not known
CAZy DB link
http://www.cazy.org/fam/GH48.html

Family 48 glycoside hydrolases are major and key components of some cellulase systems, occurring in free enzyme systems (e.g., in Thermobifida fusca), multi-functional enzymes (e.g, in Caldocellulosiruptor saccharolyticus), anaerobic fungi (e.g., Piromyces equi) and every cellulosome system thus far described. The GH48 cellulase is commonly the most abundant enzyme subunit in cellulosome-producing bacteria. Each bacterium usually contains a single gene that codes for a GH48 enzyme, although a few bacteria (e.g., and Anaerocellum thermophilum) contain two or more GH48 genes. Of the two C. thermocellum GH48 enzymes, one (Cel48S) is a dockerin-containing cellulosomal enzyme, and the other (Cel48Y) is a free, non-cellulosomal enzyme that contains a cellulose-binding CBM3.

Substrate specificities

The following activities have been reported: endo-b-1,4-glucanase, chitinase, endo-processive cellulase and cellobiohydrolase. Its preferred substrate is amorphous or crystalline cellulose over carboxymethylcellulose (CMC), and its activity is strongly inhibited by the presence of cellobiose. Although its activity on various substrates is characteristically very low, it is thought to be a critically important enzyme which imparts a major component of synergy to its cellulase system.

Kinetics and Mechanism

The glycoside hydrolases of this family includes inverting glycosidases, which preferentially attack the reducing end of the substrate [1].

The native and recombinant Cel48S from C. thermocellum displays typical characteristics of a processive exoglucanase [2], and its activity on amorphous cellulose is optimal at 70 °C and at pH 5–6.

Family 48 cellulases (i.e., CelS/S8 from C. thermocellum, Avicelase II of C. stercorarium) are stabilized at high temperatures by Ca2+ or other bivalent ions (Bronnenmeier et al., 1991; Morag et al., 1991; Kruus et al., 1995).

The Cel48F protein from Clostridium cellulolyticum has been reported (Reverbel-Leroy et al., 1997a) to be a processive endo-glucanase, which performs a processive degradation of the cellulose chain after an initial endo-attack. A two-step mechanism was proposed (parsiegla 2007 ref7), in which processive action and chain disruption occupy different subsites.

Catalytic Residues

The crystal structure of Cel48F, a cellulosome component of C. cellulolyticum, revealed the active center at the junction of the cleft and tunnel regions, where Glu55 is the proposed proton donor in the cleavage reaction, and the corresponding base was initially proposed to be either Glu44 or Asp230.( Parsiegla, 98).

The structure of the catalytic module of Cel48S of C. thermocellum showed a similar tunnel-shaped substrate-binding region formed by the alpha helices in the protein. The hydrolysis of the cellulose chain in Cel48S appeared to involve Glu87 (the equivalent of Glu55 in C. cellulolyticum Cel48F) as an acid to protonate the glycosidic oxygen atom and Tyr351 as a base to extract a proton from the nucleophilic water molecule that attacks the anomeric carbon atom.

More recent studies of Cel48F failed to unambiguously identity the catalytic base in the cleavage reaction [3].

Three-dimensional structures

Three-dimensional structures are available for two family 48 enzymes: Cel48F (from Clostridium cellulolyticum) and Cel48A (from Clostridium thermocellum). Both enzymes have an (α/α)6 barrel topology.

Family Firsts

First sterochemistry determination

Cellulomonas fimi CenE, described as an endo-β-1,4-glucanase, catalyzes the hydrolysis of cellohexaose with inversion of anomeric carbon configuration, characteristic of a single displacement reaction.

[4].

First catalytic nucleophile identification

…“Waiting patiently”… (see Parsiegla 2007)

[5].

First general acid/base residue identification

Cite some reference here, with a short (1-2 sentence) explanation [6].

First 3-D structure

The crystal structure of catalytic module of C. cellulolyticum Cel48F in complex with oligosaccharides.

First cloning and sequencing

The cel48S gene from C. thermocellum.

[7].

References

1. Katosova LK (1977). [Cytochemical characteristics of blood leukocytes during an experimental purulent-inflammatory process in rabbits]. Zh Mikrobiol Epidemiol Immunobiol. 1977(3):102-7. | Google Books | Open Library
2. Guimarães BG, Souchon H, Lytle BL, David Wu JH, and Alzari PM. (2002). The crystal structure and catalytic mechanism of cellobiohydrolase CelS, the major enzymatic component of the Clostridium thermocellum Cellulosome. J Mol Biol. 2002;320(3):587-96. DOI:10.1016/s0022-2836(02)00497-7 |
3. Parsiegla G, Juy M, Reverbel-Leroy C, Tardif C, Belaïch JP, Driguez H, and Haser R. (1998). The crystal structure of the processive endocellulase CelF of Clostridium cellulolyticum in complex with a thiooligosaccharide inhibitor at 2.0 A resolution. EMBO J. 1998;17(19):5551-62. DOI:10.1093/emboj/17.19.5551 |
8. Wang WK, Kruus K, and Wu JH. (1993). Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component. J Bacteriol. 1993;175(5):1293-302. DOI:10.1128/jb.175.5.1293-1302.1993 |
9. Guimarães BG, Souchon H, Lytle BL, David Wu JH, and Alzari PM. (2002). The crystal structure and catalytic mechanism of cellobiohydrolase CelS, the major enzymatic component of the Clostridium thermocellum Cellulosome. J Mol Biol. 2002;320(3):587-96. DOI:10.1016/s0022-2836(02)00497-7 |