The first crystal structure of a xylobiose-bound xylobiohydrolase with high functional specificity from the bacterial glycoside hydrolase family 30, subfamily 10

Abstract

Xylobiose is a prebiotic sugar that has applications in functional foods. This report describes the first X-ray crystallographic structure models of apo and xylobiose-bound forms of a xylobiohydrolase (XBH) from Acetivibrio clariflavus. This xylan-active enzyme, a member of the recently described glycoside hydrolase family 30 (GH30), subfamily 10, phylogenetic clade has been shown to strictly release xylobiose as its primary hydrolysis product. Inspection of the apo structure reveals a glycone region X₂ -binding slot. When X₂ binds, the non-reducing xylose in the -2 subsite is highly coordinated with numerous hydrogen bond contacts while contacts in the -1 subsite mostly reflect interactions typical for GH30 and enzymes in clan A of the carbohydrate-active enzymes database (CAZy). This structure provides an explanation for the high functional specificity of this new bacterial GH30 XBH subfamily.

Keywords: Acetivibrio clariflavus; GH30_10; cellulosome; glycoside hydrolase; prebiotic; xylobiohydrolase.

Fig. 1.

A phylogram showing GH30 subfamily divisions and highlighting the structurally characterized xylan-active enzymes and others used for comparisons in this research report. For this tree, all biochemically and/or structurally characterized GH30 enzymes (along with a few uncharacterized GH30s) have been included. The alignment was performed using mafftauto with structure restrains provided. This Bootstrap maximum-likelihood tree was prepared using megax. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. This analysis involved 52 amino acid sequences. There was a total of 807 positions in the final dataset. This Bootstrap consensus tree was manually rooted at the branch connecting GH30 group 1 and group 2 sequences. Red: GH30_10; green: GH30_8; blue GH30_7. Tip labels consist of the GH30 subfamily if known, PDB or UniProt accession number, organism name (first letter genus, species name), followed by the protein designation if available.

Fig. 2.

General protein fold (A) of AcXbh30A (PDB accession: 7N6H) showing the top side of the catalytic centre indicated by the juxtaposed glutamic acid amino acid side chains (upper image). The amino acids Glu168 and Glu261 (shown in pink) are known to serve as the catalytic acid–base catalyst and catalytic nucleophile, respectively [49]. Several of the barrel loop regions are indicated given their significant role in enzyme function or to guide structure visualization. A rotation of ca. ~ 135° reveals (lower image) the bottom side of the (β/α)8 + β fold and shows the double-hinge region which connects the central catalytic (β/α)₈ barrel with the side β-structure. The distribution of β-sheets in this motif is represented by the N-terminal (blue) to C-terminal (red) rainbow coloration. The double-hinge structure feature of this type occurs in several glycoside hydrolase families [2]. A secondary structure map (B) provides a two-dimensional representation of this characteristic and labels secondary structure elements important to the AcXbh30A and general GH30 protein fold. Compared to other GH30 group 2 protein structures which have a single βs1, this part of AcXbh30A is interrupted with a non-β-strand section so the naming accommodates this by designated βs1a and βs1b. The α-helical section labelled β1-α1 to indicate a α-helix-structured loop region at first glance may be considered an extension of the α1-helix. However, this specific secondary structure element is conserved in the GH30_7 but not the GH30_8 xylanases. In contrast, the two parts of the α5-helix, α5a and α5b, are observed in all GH30 group 2 structures. The α6 region is similar to that found in GH30_8 xylanases, but this same α-helix does not form in GH30_7 xylanases. Lastly, the β-hairpin structure within the β8-α8 loop is considered as such as it is not conserved in the GH30_8 xylanases. Overall, the secondary structure elements of AcXbh30A appear more like GH30_7 than GH30_8 xylanases.

Fig. 3.

Macro-structure comparison of AcXbh30A with structurally and biochemically characterized GH30_7 and 8 enzymes. A visual of just the β2-α2, β7-α7 and β8-α8 loops of AcXbh30A shown in blue, yellow and red coloration, respectively. This was overlaid with related structurally characterized GH30 xylan-active enzymes from GH30_8 (A) and GH30_7 (B–D) (all in grey). In each case, the β2-α2, β7-α7 and β8-α8 loop regions were compared to identify differential features. Comparison to the unique GH30_8 endoxylanase CaXyn30A (A, PDB accession: 5CXP) from Clostridium acetobutylicum shows a diminutive form of both of these loop regions. Overlay comparison with the GH30_7 endoxylanase TcXyn30C (B, PDB accession: 6M5Z) from Talaromyces cellulolyticus shows a still minor β2-α2 loop with a slightly larger β8-α8 loop. Most comparable the two dual-function GH30_7 xylobiohydrolase/endoxylanase functioning enzymes TcXyn30B (C, PDB accession: 6KRN) from T. cellulolyticus and TtXyn30A (D, PDB accession: 7O0E) from Thermothelomyces thermophila shows only a slightly smaller β2-α2 loop and a β8-α8 loop which, while still smaller, is more significant than the GH30_8 endoxylanase and the GH30_7 TcXyn30C.

Fig. 4.

Inconsistent coordination observed for the two 2²-(4-O-methyl-α-D-glucuronosyl)-xylobiose (U^4m2X) bound GH30_7 GXN/XBH dual-function enzymes. TcXyn30B (green, PDB accession: 6KRN) and TtXyn30A (red-brown, PDB accession: 7O0E) share nearly 45% amino acid sequence identity. The β2-α2, β7-α7 and β8-α8 loop regions (A) primarily involved in ligand coordination align well for these two similarly functioning enzymes. Further analysis of the protein structure superposition of these two GH30_7 enzymes shows a number of conserved amino acid side chains in alternative positions including the ‘eukaryotic arginine’ (B) thought to have a role in GA coordination. Although these enzymes are thought to function similarly, the hydrogen bonding differences between these proteins and their crystallographically modelled ligand clearly shows differences in their ‘specific’ contacts.

Fig. 5.

Structure analysis of the glycone xylobiose-binding slot. Surface representation (A) of the AcXbh30A glycone region depicting the crystallographically determined xylobiose positioned in a slot that is formed from the β2-α2 (blue), β4-α4 (green), β7-α7 (orange) and β8-α8 (red) loops. An electron density map visual (B) of the xylobiose-binding region intended to represent the crystallographic data quality. The map around the X₂ is from a CCP4 Omit program map and the density around the amino acid side chains is from the refinement-generated 2Fo-fc map. A representation (C) of the amino acid side chains establishing polar or van der Waals contacts with the X₂ and therefore shaping the glycone xylobiose-binding slot and revealing the specificity for the non-reducing terminal xylobiose activity. Lastly, a ligplot+ plot (D) [42] showing all the predicted molecular contacts involved in X₂ positioning into the glycone region.

Fig. 6.

Analysis of the Met265 position in AcXbh30A and comparison with GH30_7 homologs. Comparison of the position of Met265 (A) in the apo form (light grey, PDB accession: 7N6H) and X₂-bound form (dark grey, PDB accession: 7N6O). It is seen that the ε-C repositions upon substrate binding and result in an orientation that may allow interaction with the +1 subsite xylose. Also shown in the conserved leucine from GH30_7 xylan-active enzymes represented here by Leu288 from TcXyn30C (cyan, PDB accession: 6M5Z). In this, the δ1-C resides in nearly the same position as the Met265 δ-S from AcXbh30A. In the apo form, the position of ε-C of Met265 would clash with a −1 subsite xylose being just 1.9 Å away from the C5 of the xylose ring (B), and while oriented towards the +1 subsite following ligand binding, it would seem likely to engage through van der Waals interactions. More importantly, δ-S in the X₂-bound form, at 3.7 Å away, looks likely to interact specifically [55] with the endocyclic oxygen of the −1 subsite xylose. Further consideration of this region of AcXbh30A (C) with the aglycone X₂ modelled from a simple structure alignment with the GH30–8 glucuronoxylanase CtXyn30A (PDB accession: 5A6M, see Fig. S2) shows that a second methionine, Met212, may interact with the endocyclic oxygen of the +1 subsite xylose. Comparisons of AcXbh30A (D) (ac, rainbow) with the dual-function GH30_7 GXN/XBH, TcXyn30B (Tc, pink) and TtXyn30A (Tt, cyan) and the homology model of the GH30_7 XBH AaXyn30A (aa, grey). The position of the −2 subsite xylose is shown (AcXbh30A, green; TcXyn30B, purple, TtXyn30A, aquamarine).

Fig. 7.

Comparison of GH30 subfamily 7, 8 and 10 substrate-binding cleft aglycone regions. The aglycone region of AcXbh30A (A; PDB accession 7N6O, white) showing for perspective, the two catalytic glutamates and Met265 which are thought to interact with both the −1 (glycone) and +1 (aglycone) subsite xylose residues. For comparison, the aglycone region of three GH30_8 and three GH30_7 structurally characterized enzymes is shown (B, C). The GH30_8 enzymes (B) include the non-canonical functioning, GA-independent endoxylanase CaXyn30A (PDB accession: 5CXP; green) and the two canonical functioning GA appendage-dependent endoxylanases BsXynC (PDB accession: 3KL5; olive) and CtXyn30A (PDB accession: 5A6M; light green). Amino acid labels correspond to the CaXyn30A structure and where not fully conserved, following a forward slash other representative amino acids are listed. For the GH30_7 enzymes (C), TcXyn30B (PDB accession: 6KRN; blue), TcXyn30C (PDB accession: 6M5Z; purple) and TtXyn30A (PDB accession: 7O0E; dark blue) are shown. Amino acid labels correspond to TcXyn30B and where not fully conserved, following a forward slash other representative amino acids are listed. Overlaying all structures from A to C (D), the positions are labelled with the list of amino acids represented in that group. Significant diversity exists in the aglycone region of these GH30 group 2 xylan-active enzymes.