Journal:Acta Cryst D:S2059798319007113

Crystal Structure Determination of Pseudomonas stutzeri A1501 endoglucanase Cel5A

Raphael Dutoit, Maud Delsaute, Laetitia Collet, Corinne Vander Wauven, Dany Van Elder, Renaud Berlemont, Aurore Richel, Moreno Galleni and Cedric Bauvois ^[1]

Molecular Tour

Some glycoside hydrolases (GH) possess the inherent ability to transglycosylate via the retaining double displacement mechanism. During transglycosylation, a carbohydrate hydroxyl group acts as an acceptor instead of water in hydrolysis. Such moonlighting GHs are often referred as transglycosylases. However, they are more closely related to hydrolytic enzymes of the same GH family than to transglycosylases of other families. This would indicate that subtle molecular adjustments rather than large modifications allow transglycosylation. These molecular determinants can be studied by comparing transglycosylases with pure hydrolytic GH of the same sub-family. Several examples of transglycosylase have been reported in the GH5 family. This family is one of the largest GH families and includes enzymes with various activities and specificities (like endo-1,4-b-glucanases, b-mannanases, exo-1,3-b-glucanases, endo-1,4-b-xylanases, lichenases, etc.).

We previously identified RBcel1 (PDB entry 4ee9), an endo-1,4-b-glucanase of the GH5_5 subfamily, as able to catalyze the polymerization of cello-oligosaccharides. Its closest sequence homologue is Ps_Cel5A, a putative endoglucanase of Pseudomonas stutzeri. Its activity was characterized revealing that Ps_Cel5A is a moonlighting GH able to hydrolyze carboxymethylcellulose and to synthetize cellotetraose and cellohexaose from cellotriose and cellopentaose, respectively. The (PDB entry 4lx4) and in (PDB entry 6r2j). (PDB entry 6r2j). Cellobiose is positioned in the acceptor binding site (+1 and +2 subsites) while Tris occupies the -1 subsite (donor). This scene illustrate the structural adaption possibly linked to transglycosylation. Both Ps_Cel5A and RBcel1 display a slight difference in the acceptor binding site compared to other GH5_5 members described as pure hydrolytic enzymes (i.e. Thermoascus aurantiacus Cel5A, Hypocrea jecorina Cel5A, Aspergillus niger Cel5A, and Ganoderma lucidum Cel5A).

. A non-conserved arginine residue (, respectively) is found in the acceptor binding site. . Such an interaction could increase the acceptor binding affinity and, hence, favor transglycosylation. This structural feature is not observed in other GH5_5 members. Remarkably, the presence of arginine residue in the acceptor binding site does not occur in a conserved position in Ps_Cel5A and Rbcel1. Indeed, His184 is found in Ps_Cel5A at the equivalent position to Arg176. Likewise, Ser170 is found in RBcel1 at the equivalent position to Arg178, indicating a possible convergent evolution. It is worth noting that a similar adaptation of the acceptor binding site has been reported for several b-mannanases of the GH5_7 subfamily. In these enzymes, an arginine residue plays a key role in the acceptor sugar binding, driving transglycosylation.

PDB reference: Crystal Structure of Pseudomonas stutzeri endoglucanase Cel5A in complex with cellobiose 6r2j.

References

1. ↑ Dutoit R, Delsaute M, Collet L, Vander Wauven C, Van Elder D, Berlemont R, Richel A, Galleni M, Bauvois C. Crystal structure determination of Pseudomonas stutzeri A1501 endoglucanase Cel5A: the search for a molecular basis for glycosynthesis in GH5_5 enzymes. Acta Crystallogr D Struct Biol. 2019 Jun 1;75(Pt 6):605-615. doi:, 10.1107/S2059798319007113. Epub 2019 Jun 6. PMID:31205022 doi:http://dx.doi.org/10.1107/S2059798319007113