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.