A New Group of Modular Xylanases in Glycoside Hydrolase Family 8 from Marine Bacteria

Abstract

Xylanases play a crucial role in the degradation of xylan in both terrestrial and marine environments. The endoxylanase XynB from the marine bacterium Glaciecola mesophila KMM 241 is a modular enzyme comprising a long N-terminal domain (NTD) (E44 to T562) with xylan-binding ability and a catalytic domain (CD) (T563 to E912) of glycoside hydrolase family 8 (GH8). In this study, the long NTD is confirmed to contain three different functional regions, which are NTD1 (E44 to D136), NTD2 (Y137 to A193), and NTD3 (L194 to T562). NTD1, mainly composed of eight β-strands, functions as a new type of carbohydrate-binding module (CBM), which has xylan-binding ability but no sequence similarity to any known CBM. NTD2, mainly forming two α-helices, contains one of the α-helices of the catalytic domain's (α/α)₆ barrel and therefore is essential for the activity of XynB, although it is far away from the catalytic domain in sequence. NTD3, next to the catalytic domain in sequence, is shown to be helpful in maintaining the thermostability of XynB. Thus, XynB represents a kind of xylanase with a new domain architecture. There are four other predicted glycoside hydrolase sequences with the same domain architecture and high sequence identity (≥80%) with XynB, all of which are from marine bacteria. Phylogenetic analysis shows that XynB and these homologs form a new group in GH8, representing a new class of marine bacterial xylanases. Our results shed light on xylanases, especially marine xylanases.IMPORTANCE Xylanases play a crucial role in natural xylan degradation and have been extensively used in industries such as food processing, animal feed, and kraft pulp biobleaching. Some marine bacteria have been found to secrete xylanases. Characterization of novel xylanases from marine bacteria has significance for both the clarification of xylan degradation mechanisms in the sea and the development of new enzymes for industrial application. With G. mesophila XynB as a representative, this study reveals a new group of the GH8 xylanases from marine bacteria, which have a distinct domain architecture and contain a novel carbohydrate-binding module. Thus, this study offers new knowledge on marine xylanases.

Keywords: N-terminal domain; carbohydrate-binding module; catalysis; glycoside hydrolase family 8; modular xylanase.

FIG 1

Alignment of the sequences of XynB and two other reported GH8 xylanases. The two reported GH8 xylanases are the endoxylanase PhXyl from Pseudoalteromonas haloplanktis (PDB accession number 1H13) and the reducing-end xylose-releasing exo-oligoxylanase BhRex from Bacillus halodurans (PDB accession number 1WU4). PhXyl and BhRex are both single-domain enzymes containing only a catalytic domain. Using ESPript, secondary structures of BhRex are shown above the alignment, and secondary structures of PhXyl are shown below the alignment. Helices are indicated by springs, strands are indicated by arrows, turns are indicated by TT, and 3₁₀-helices are indicated by η. Identical residues are shown in white on a red background, and similar residues are shown in bold red. Solid circles indicate residues (E586, D646, and D786) crucial for the catalytic activity of GH8 xylanases. Solid triangles indicate selected conserved residues (Y159, N161, E165, and F186) in NTD2 of XynB. The different regions in the NTD of XynB are also shown.

FIG 2

Schematic diagram of the domain architecture of XynB and its mutants. The specific activities of XynB and its mutants toward 30 mg/ml arabinoxylan or beechwood xylan were determined at 40°C and pH 7.0. The data shown in the graph are from triplicate experiments (means ± standard deviations [SD]). ND indicates that enzyme activity was not detectable.

FIG 3

SDS-PAGE analysis of the abilities of WT XynB and its mutants to bind to insoluble beechwood xylan. Bovine serum albumin (BSA) (0.1 mg) was used as a negative control. Lane C, total proteins (control); lane 1, unbound proteins; lane 2, proteins in the wash buffer; lane 3, bound proteins. The percentages at the bottom of the gel are the densitometric ratios of each band compared with that of the control band, which are means ± SD of data from triplicate experiments. The data shown are representative of results of triplicate experiments.

FIG 4

Structural model of mutant NTD2-CD. (A) Modeled structure of mutant NTD2-CD. The structure was modeled using the structure of the endoxylanase PhXyl (PDB accession number 1H13) as the template. NTD2 is shown in orange, and the catalytic domain is shown in cyan. Catalytic residues E586, D646, and D786 of XynB are shown as magenta sticks. Conserved residues Y159, N161, E165, and F186 in NTD2 are also shown. (B) Crystal structure of the D144A mutant of PhXyl in complex with xylopentaose (PDB accession number 2B4F). PhXyl is a single-domain enzyme containing only a catalytic domain. The region in PhXyl sharing similarity to NTD2 of XynB is shown in pink, and the other region is shown in green. Catalytic residues E78, D144 (replaced by Ala in the D144A mutant), and D281 are shown as magenta sticks, and xylopentaose is shown as yellow sticks.

FIG 5

Effects of temperature, NaCl, and pH on the activities and stabilities of WT XynB and its mutants. (A) Effect of temperature on the activities of WT XynB and its mutants. (B) Effect of temperature on the stabilities of WT XynB and its mutants. The enzyme was incubated at 35°C for different periods. Residual activity was measured under optimal conditions. (C) Effect of NaCl on the activities of WT XynB and its mutants. (D) Effect of NaCl on the stabilities of WT XynB and its mutants. The enzyme was incubated in buffers containing different concentrations of NaCl (0 to 4 M) at 4°C for 1 h. Residual activity was measured under optimal conditions. (E) Effects of pH on the activities (left) and stabilities (right) of WT XynB and its mutants. To determine the effect of pH on enzyme stability, the enzymes were incubated at 4°C for 1 h in buffers ranging from pH 4.0 to 10.0. Residual activity was measured under optimal conditions. The activities of WT XynB, XynBΔNTD1, and NTD2-CD at 40°C (A), at 0 min (B), and with 0 M NaCl (C and D) and the maximum activities (E) were taken as 100%, respectively. The graphs show data from triplicate experiments (means ± SD).

FIG 6

Phylogenetic tree of XynB and reported GH8 xylanases. The tree was built by using the neighbor-joining method with a Jones-Taylor-Thornton (JTT) matrix-based model using 297 amino acid positions. Bootstrap analysis of 1,000 replicates was conducted, and values above 50% are shown. The scale for the branch length is shown below the tree. Endoglucanases and chitosanases from GH8 were used as an outgroup.