Chapter 1. General Introduction 1
2.2. Material and methods
2.3.3. Substrate recognition by CtAbf43A
Fig 2.4. Structure-based sequence alignment of CtAbf43A (PDB ID 5A8C) from C.
thermocellum, with other members of GH43_16 sub-family named by accession numbers and B. subtilis BsAXH-m2,3 arabinofuranosidase (PDB ID 3C7E). The alignment includes the sequences of Cellvibrio japonicus highly specific α-1,2-L-arabinofuranosidase (PDB ID 3QEF) and the ruminal GH43 arabinofuranosidase of Butyrivibrio proteoclasticus (PDB ID 4NOV), which are members of GH43_29 sub-family. Blue filled stars represent conserved key catalytic residues. The regions contributing to the five structural blades of CtAbf43A are represented.
Fig. 2.5. The active site of CtAbf43A with a putative superposed xylotetraose ligand from B. subtilis BsAXH-m2,3 complex (PDB ID 3C7G). The active site subsites are labeled. The β-1,4 tetrasaccharide is depicted in yellow in a ball
& stick representation while the Tris molecule in purple was found at subsite -1 in CtAbf43A. The side chains of relevant residues are shown:
related catalytic residues in ball & stick representation with green labels;
polar contact residues in stick representation with blue labels and hydrophobic/stacking residues in wire representation with yellow labels.
XYP: xylotetraose.
Superposition of CtAbf43A with BsAXH-m2,3 in complex with xylotetraose (Vandermarliere et al., 2009) revealed the structural properties of the various subsites of GH43_16 enzymes that modulate arabinoxylan specificity (Fig. 2.5). Thus, at the +3R subsite Ser-268 is predicted to make several hydrogen bonds with xylose O-2 and O-3. At subsite, +2R Phe-234 makes strong hydrophobic interactions with xylose and Asn-269, replaced by Gly-289 in the Bacillus enzyme, could make polar contacts with the sugar. At subsite +1, Glu-215, the putative catalytic acid, makes two strong hydrogen bonds with xylose O-2 and O-3, which may comprise the scissile glycosidic bond, and the Nδ2 of Asn-270 is within hydrogen-bonding distance of O-2 of the sugar
moiety. Phe-214 provides a hydrophobic environment to stack xylose at subsite +1 and may also create an apolar context that contributes to elevate the pKa of the acidic residue. Significantly, at subsite +1, CtAbf43A contains a tyrosine residue, Tyr-70, which is not present in BsAXH-m2,3. In the Bacillus enzyme this residue is replaced by phenylalanine. The Oη of Tyr-70 is at hydrogen bond distance to O-2 and O-3 of the xylose residue accommodated at subsite +1, the two putative scissile bonds. This suggests an important role of Tyr-70 in the stabilization of the substrate in the proximity of the catalytic site.
Similarly, to subsite +2R, in subsite +2NR binding is solely based in hydrophobic stacking interactions as the xylose residue is sandwiched between the aromatic residues Trp-163 and Trp-106. As described above, CtAbf43A may contain a +3NR subsite that may accommodate the xylose backbone or eventually make productive interactions with it. Although this subsite could be solvent-exposed, Oδ1 and Nδ2 of Asn-126 could turn the +3NR subsite functional through the establishment of polar contacts. With the exception of the replacement of Tyr-70 in CtAbf43A by Phe- 56 in B. subtilis (BsAXH-m2,3) at the +1 subsite, and Asn-269 by Gly-289 at the +2R subsite, the residues that participate in recognition of the arabinoxylan backbone are remarkably conserved in the two GH43_16 enzymes. Inspection of the primary sequence of GH43_16 members, suggest that this conservation spans the entire sub- family.
The catalytic pocket that houses the -1 subsite reveals several conserved residues in GH43 enzymes which may contribute to the fine-tuning of substrate binding and transition state stabilization. Thus, Asp-43, the general base, Glu-215, the general acid and Asp-166, the third catalytic residue that is believed to modulate the pKa of the
catalytic acid as well as orientating the catalytic acid and the substrate, are conserved in GH43 enzymes (Vandermarliere et al., 2009; Cartmell et al., 2011). These residues are located on the most internal strands of blades 1, 4 and 3, respectively and are conserved in GH43 family (Fig. 2.4). As the catalytic base activates a water molecule, which attacks the anomeric C atom from one side, the catalytic acid donates a proton to the leaving group on the other side, cleaving the glycosidic bond between the two sugar moieties and leading to an inversion of the configuration of the anomeric C atom. The catalytic role of Asp-43 and Glu-215 was confirmed by the observation that CtAbf43A mutant derivatives Asp43Ala and Glu215Ala are inactive. The inability of the two mutant derivatives to perform catalysis was confirmed by assaying the enzymes with both natural and artificial substrates and by measuring product release by spectrophotometrically. Trp-106 and Ile-72 are highly conserved in several GH43 enzymes and provide the hydrophobic platform that stacks the sugar rings at the -1 subsite. In addition, Arg-303, His-287 and His-272 are mostly unchanged in GH43 enzymes and may also contribute polar contacts with the arabinose housed at the enzyme active site. Also, owing to their basic nature, these three residues may contribute to decrease the pKa of the catalytic base, Asp-43. The structure of CtAbf43A revealed that the -1 subsite houses a molecule of 2-amino-2-hydroxymethyl-propane- 1,3-diol that is most likely occupying the position of arabinose during catalysis. A close inspection of CtAbf43A -1 subsite revealed that this molecule establishes several hydrogen bonds with the catalytic residues Asp-43, Asp-166 and Glu-215, but also with Arg-303, Tyr-70 and His-272, confirming the putative involvement of these later amino acids in substrate recognition (Fig. 2.6).
Fig. 2.6. Interactions established between a molecule of Tris, identified at subsite -1, and CtAbf43A.
Recently, it was suggested that His-272 is involved in catalysis via an electron- withdrawing pathway between the divalent calcium ion and the catalytic acid Glu215, thus helping to modulate the pKa of the acidic residue (Till et al., 2014). The N∊2 of His-272 of CtAbf43A participates in the coordination of the central calciumion (Fig.
2.3). In CtAbf43A, Nδ1 of His-272 is hydrogen-bonded to the Tris molecule found at the -1 subsite which subsequently binds to Oδ1 of Glu-215. This electron-withdrawing pathway observed in CtAbf43A structure supports the suggestion that the calcium ion and His-272 are positioned in a way that could exert an effect on the pKa of Glu-215 (Till et al., 2014). CtAbf43A calcium ion interacts with seven ligands in pentagonal bi- pyramidal coordination previously observed in other GH43 enzymes. The axial positions are occupied by N∊2 of His-272 described above and one water molecule. The five equatorial ligands are all water molecules and are positioned midway between two of the blades of the propeller allowing each water molecule to hydrogen-bond to either
proline or the preceding residue located in the loops. In CtAbf43A these prolines are located near the catalytic residues. One of these prolines, Pro-108, is located at the end of the loop that terminates at the first β-strand of the first blade located next to Asp-43, the general base. Pro-108 participates in hydrogen bonding to one of the equatorial waters and belongs to the highly conserved motif “WAP” of GH43 domains (Vandermarliere et al., 2009), of which the tryptophan, Trp-106, was suggested to form hydrophobic stacking interactions with the substrate.