Difference between revisions of "Glycoside Hydrolase Family 48"

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

Glycoside Hydrolase Family GH48
Clan	GH-M
Mechanism	inverting
Active site residues	Proton donor: known; Nucleophile: unknown
CAZy DB link
https://www.cazy.org/GH48.html

Substrate specificities

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 Caldicellulosiruptor 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., Clostridium thermocellum 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.

The following activities have been reported: endo-β-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 are 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 [3, 4, 5].

The Cel48F protein from C. cellulolyticum has been reported [6] 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 [7], 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 [8].

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 [7].

A recent experimental study in Thermobifida fusca Cel48A confirmed that aspartic acid (Asp225) is the catalytic base in family 48 glycoside hydrolases [9]. This residue is equivalent to D230 of C. cellulolyticum Cel48F and D255 of C. thermocellum Cel48S. In this study, site-directed mutagenesis demonstrated that the D225E mutation retained partial activity on soluble and insoluble substrates. Azide rescue hydrolysis assays showed that the D225G mutation restored its activity with added azide.

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.

3D structures of GH48 proteins (click images for large versions)
PDB ID 1fae from "Crystal structure of the cellulase CEL48F from C. cellulolyticum in complex with cellobiose" [10].
PDB ID 1l1y and 1l2a from "The Crystal Structure and Catalytic Mechanism of Cellobiohydrolase CelS, the Major Enzymatic Component of the Clostridium thermocellum cellulosome" [11], in complex with cellohexaose and cellobiose.

3D structures of Cel48F in complex with different ligands are also available:

with cellotetraose (1f9d)
with the thio-oligosaccharide inhibitor PIPS-IG3 (1f9o)
with cellobiose (1fae)
with cellobiitol (1fbo)
with cellohexaose (1fbw)
with a thio-oligosaccharide (1g9j)
mutant E55Q with a thio-oligosaccharide (2qno)

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 [12].

First catalytic nucleophile identification: Asp225 was experimentally shown to be the catalytic base in T. fusca Cel48A [9].

First general acid/base residue identification: Glu was the proposed proton donor in the cleavage reaction [9].

First 3-D structure: The crystal structure of catalytic module of C. cellulolyticum Cel48F in complex with oligosaccharides [7].

First cloning and sequencing: The cel48S gene from C. thermocellum [13].

References

All Medline abstracts: PubMed

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 |{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
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-| colspan="2" |http://www.cazy.org/fam/GH48.html
+| colspan="2" |{{CAZyDBlink}}GH48.html
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 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 <cite>Parsiegla2008</cite>.
-A recent experimental study in ''Thermobifida fusca'' Cel48A confirmed that aspartic acid (Asp225) is the catalytic base in family 48 glycosyl hydrolases <cite>Kostylev2011</cite>. This residue is equivalent to D230 of ''C. cellulolyticum'' Cel48F and D255 of ''C. thermocellum'' Cel48S. In this study, site-directed mutagenesis demonstrated that the D225E mutation retained partial activity on soluble and insoluble substrates. Azide rescue hydrolysis assays showed that the D225G mutation restored its activity with added azide.
+A recent experimental study in ''Thermobifida fusca'' Cel48A confirmed that aspartic acid (Asp225) is the catalytic base in family 48 glycoside hydrolases <cite>Kostylev2011</cite>. This residue is equivalent to D230 of ''C. cellulolyticum'' Cel48F and D255 of ''C. thermocellum'' Cel48S. In this study, site-directed mutagenesis demonstrated that the D225E mutation retained partial activity on soluble and insoluble substrates. Azide rescue hydrolysis assays showed that the D225G mutation restored its activity with added azide.
 == Three-dimensional structures ==