3.2.3. Exocellulases
Exoglucanases or cellobiohydrolases (CBH) (EC 3.2.1.74; 1,4-β-D-glucan-glucanhydrolase) catalyze the successive hydrolysis of residues from the reducing and non-reducing ends of the cellulose polysaccharide, releasing cellobiose molecules as main product of the reaction 38. These enzymes account for 40 to 70% of the total component of the cellulase system, and are able to hydrolyze crystalline cellulose.
An excellent candidate for use as a bait to explore algal genomes is the GH10 from Cellulomonas fimi (Cex- P07986 Uniprot). High resolution crystal structures are available and there is a large literature on the kinetic characterization of the enzyme and the identification of amino acid residues important to the mechanism of catalysis 39. Among all the Scenedesmaceae genomes studied we found at least eleven enzymes, all of them putative GH10 bifunctional cellulase/xylanase proteins (Figure 12A).
These enzymes are monomeric proteins with a molecular mass ranging from 50 to 65 kDa, although there are smaller variants (41.5 kDa) in some fungi, such as Sclerotium rolfsii 40. The calculated molecular mass of five of the eleven algal proteins studied were higher (between 70 and 100kDa), while in the other six enzymes is within the expected range (table 2).
In general, most of the proteins containing GH10 domains show a structure that matches an eightfold α/ β-barrel with a profound channel in the center 41. However, it has been proposed that the exocellulases from the GH10 family form a transient tunnel by the extension of some loop regions upon substrate binding42.
It has been reported that GH10 enzymes present a double-displacement ’retaining’ hydrolysis mechanism, where one catalytic residue acts a nucleophile and the other acts as a general acid/base43. The catalytic nucleophile in Cex is Glu274 and the putative acid/base catalyst is Glu168 44. As shown in figure 12B, both residues are full conserved in the algal protein sequences. However, they were replaced by an Asp and Ile residues in the Scequ2611|547 protein.
We also performed a phylogenetic tree for the GH10 exoglucanase (Figure 13) from the alignment of the amino acid sequences fromScenedesmus GH10 enzymes together with homologous sequences from invertebrates, fungi, plants and bacteria. The branches distribution suggests the GH10 analyzed proteins are evolutionarily closer to those of other microalgae and higher plants.
In addition, we built a sequence homology model of the Sceob1|4623 exocellulase using RaptorX Contact Prediction is shown in Figure 14. The superposition with Cellulomonas fimiexocellulase crystal structure (PBD code: 1EXG) shows a spatial location conservation of the algal protein residues Glu236 and Glu338 with respect to catalytic residues from the bacterial protein, also suggesting the conservation of the catalytic site.