6-Hydroxydopamine hydrobromide Docking experiments showed th
Docking experiments showed that Me-MeGlcA3Xyl4 and MeGlc3Xyl4 were bound to EcXyn30A and the R293A variant in the same way as MeGlcA3Xyl4, having MeGlcA or its modified forms accommodated in the -2b subsite. All ligands were coordinated by the same 6-Hydroxydopamine hydrobromide and no new interactions were observed. The only difference was the contribution of particular amino acids to the total binding energy which was highest for EcXyn30A–MeGlcA3Xyl4 complex. The decrease in total binding energies calculated for the docked Me-MeGlcA3Xyl4–EcXyn30A and MeGlc3Xyl4–EcXyn30A models was mostly due to a reduced interaction of R293 confirming that its interaction with MeGlcA carboxylate is important for the natural substrate binding. The proposed identical way of productive binding of all three substrates is supported by the fact that EcXyn30A, as well as the R293A variant, produced from all three polymeric substrates (GX, GXE and GXR) series of xylooligosaccharides having a side chain on the second xylose from the reducing end as a result of the accommodation of the branched residue in the -2a subsite. The docking experiments suggested that the difference in catalytic efficiencies of EcXyn30A and the R293A variant on GX, GXE and GXR is not a consequence of different binding of the substrates but rather a result of a change in binding energies represented by increased Km which may be combined with decreased kcat. High decrease in kcat was observed only for the native enzyme with the modified substrates which lack a counterpart for making the ionic interaction with Arg293. Guanidino group of the arginine, that is conserved in GH30_8 glucuronoxylanases, has itself a strong propensity to form a bidentate interaction. Several anions, such as phosphate (PDB ID: 5A6M), sulphate (PDB ID: 4UQB), tartarate (PDB ID: 4UQC) and malonate (PDB IDs: 4CKQ, 3KL0) have been observed to interact with the corresponding arginine residue in carbohydrate ligand-free structures of several GH30 glucuronoxylanases [20,27] including EcXyn30A with bound acetate (PDB ID: 1NOF) . All hydrolysis experiments reported in this study were performed in acetate buffer. Therefore, we can expect acetate ion to interact with Arg293. GX and aldouronic acids (e.g. MeGlcA3Xyl4) are apparently able to displace the acetate. Such a displacement is likely to be facilitated by other enzyme-carbohydrate interactions in −1, -2a and particularly -2b subsites. On the other hand, the acetate may remain bound to Arg293 when a substrate without negatively charged group is to be processed. The acetate thus impairs productive binding of the modified glucuronoxylans that is reflected in the high decrease in kcat for the native enzyme. Of course, the interference of the acetate is not expected in the R293A variant due to a loss of guanidino group in the modified enzyme. There is only one report on a GH30 xylanase that behaves as MeGlcA-dependent glucuronoxylanase but does not contain arginine residue corresponding to the Arg293 of EcXyn30A. It is XynVI of Trichoderma reesei belonging to GH30_7 subfamily together with other fungal xylanases . This subfamily includes also XynIV from T. reesei  and XYLD from Bispora sp. MEY-1  which are not MeGlcA-dependent. Alignment of amino acid sequences of fungal and bacterial GH30 xylanases, grouped to GH30_7 and GH30_8 subfamilies, respectively, revealed several conserved amino acids responsible for high glucuronoxylan specificity of bacterial xylanases which are not present in fungal xylanases [10,13]. Unfortunately, the structure of any fungal GH30 xylanase has not been solved yet, therefore, it is difficult to predict precisely a function of a particular amino acid and explain catalytic properties of fungal GH30_7 xylanases on the structural basis. An opposite example is Xyn30A from Clostridium papyrosolvens. As a bacterial xylanase it belongs to GH30_8 subfamily, however, it does not exhibit specificity for MeGlcA appendage . The reason is a difference in the structure of substrate binding site of CpXyn30A which does not contain analogs to R272/R293, Y274/Y295 and S235/S258, all described to interact with MeGlcA in B. subtilis XynC and EcXyn30A, respectively [19,20], and all being conserved in other GH30_8 glucuronoxylanases.