A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity

Abstract

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyrosolvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark β8-α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved β8-α8 loop region of these enzymes influences xylan substrate specificity but not necessarily β-1,4-xylanase function.

Keywords: glycoside hydrolase family 30; low-value biomass; wildfire; xylanase.

Figure 1

Sequence analysis of CpXyn30A. (a) An alignment with conserved secondary-structure elements of the β7–α7 and β8–α8 loop regions representing the area of GA coordination in the characterized GH30-8 enzymes (BsXynC, Bs_Q45070; EcXynA, Ec_Q46961). The specific regions which contain the amino acids which directly coordinate the GA are boxed in blue and the same regions from CpXyn30A are in red. (b) The domain architecture of CpXyn30A showing the GH30 catalytic module followed by a family 6 CBM and two dockerin domains presumably for inclusion in a cellulosome complex. (c) Phylogram depicting the distribution of CpXyn30A relative to homologous GH30-8 Gram-positive and Gram-negative derived enzymes rooted to a GH30-1 enzyme. Sequences are identified through their unique UniProt accession number preceded by the initials of the bacterium. Other sequences used in this comparison included Cs_M1MAL3 from C. saccharoperbutylacetonicum, Ra_E6UFE8 from Ruminococcus albus, Cj_B3PEH7 from Cellvibrio japonicus, Xp_F0BZ40 from Xanthomonas perforans and the GH30-1 subfamily enzyme Hs_P04062 from Homo sapiens used as an outgroup for the phylogenetic analysis.

Figure 2

Comparison of CpXyn30A with similar GH30-8 enzymes. (a) Structural alignment of CpXyn30A (green) and BsXynC (cyan) visualized from the bottom showing the distinctive dual linker region which connects the side β-domain to the catalytic (α/β)₈-barrel. (b) An extended α-helix associated with the α2 helix in CpXyn30A provides two additional stabilizing contacts not found in the other structurally characterized GH30-8 enzymes BsXynC or EcXynA (only BsXynC is shown in the figure for clarity). The one major interaction which is conserved involves the perpendicular ring stacking between Trp66 (Trp63 in BsXynC) and Tyr124 (Phe121 in BsXynC). This region of CpXyn30A additionally benefits from a similar perpendicular ring stacking between Trp58 and Tyr120 and also a hydrogen bond between Asp56 at the top of the loop and Ser93 extending down from the β3–α3 loop region. (c) The β3–α3 and β4–α4 loop region of CpXyn30A is homologous to that from other Gram-positive GH30-8 structures aligning closely with BsXynC but not with the Gram-negative model GH30-8 xylanase EcXynA (magenta).

Figure 3

Differences between the β7–α7 and β8–α8 loop regions of CpXyn30A (green) and BsXynC (cyan) would not allow the coordination of GA residues. (a) Coordination of aldotriuronate into the BsXynC (PDB entry 3kl5) active site shows numerous contacts for which there is no equivalent interaction in the CpXyn30A structure. (b) Structure alignment of CpXyn30A and BsXynC shows the similar C^α trace between the two enzymes, with the alternative amino-acid side chains identified by CpXyn30A/BsXynC amino-acid numbering.

Figure 4

TLC analysis of CpXyn30A and BsXynC processing of (a) polymeric xylans and (b) xylooligosaccharides. Standards consist of X₁–X₅ lanes and lanes corresponding to the aldouronate series of sugars. Lanes are designated with times in minutes, where O/N indicates that the reaction was allowed to proceed overnight.

Figure 5

HPLC analysis of xylooligosaccharide processing by CpXyn30A. A digestion of X₆ (red) by CpXyn30A after 12 min (blue) showing the early reaction build-up of larger xylooligosaccharides. The inset describes the general course of a transglycosylation event in comparison to the typical hydrolytic event.