Glycoside hydrolase from the GH76 family indicates that marine Salegentibacter sp. Hel_I_6 consumes alpha-mannan from fungi

Abstract

Microbial glycan degradation is essential to global carbon cycling. The marine bacterium Salegentibacter sp. Hel_I_6 (Bacteroidota) isolated from seawater off Helgoland island (North Sea) contains an α-mannan inducible gene cluster with a GH76 family endo-α-1,6-mannanase (ShGH76). This cluster is related to genetic loci employed by human gut bacteria to digest fungal α-mannan. Metagenomes from the Hel_I_6 isolation site revealed increasing GH76 gene frequencies in free-living bacteria during microalgae blooms, suggesting degradation of α-1,6-mannans from fungi. Recombinant ShGH76 protein activity assays with yeast α-mannan and synthetic oligomannans showed endo-α-1,6-mannanase activity. Resolved structures of apo-ShGH76 (2.0 Å) and of mutants co-crystalized with fungal mannan-mimicking α-1,6-mannotetrose (1.90 Å) and α-1,6-mannotriose (1.47 Å) retained the canonical (α/α)₆ fold, despite low identities with sequences of known GH76 structures (GH76s from gut bacteria: <27%). The apo-form active site differed from those known from gut bacteria, and co-crystallizations revealed a kinked oligomannan conformation. Co-crystallizations also revealed precise molecular-scale interactions of ShGH76 with fungal mannan-mimicking oligomannans, indicating adaptation to this particular type of substrate. Our data hence suggest presence of yet unknown fungal α-1,6-mannans in marine ecosystems, in particular during microalgal blooms.

Fig. 1. Gene synteny of GH76-containing PULs in Salegentibacter strains.

The asterisk marks remnants of a former transposase gene.

Fig. 2. Frequencies of GH76 and GH92 genes in metagenomes sampled from the Salegentibacter sp. Hel_I_6 isolation site during spring phytoplankton blooms in the years 2010, 2011, 2012, 2016 and 2018.

Samples were taken for free-living bacteria (0.2–3 µm), two size fractions of particle-attached bacteria (3–10 µm; >10 µm) and from sediments (Sed.). Frequencies are expressed as reads per kilobase million (RPKM).

Fig. 3. Phylogenetic tree of GH76 protein sequences from different genomes and metagenomes: 8 from Salegentibacter species, 11 from the CAZyme database, 64 from the Hel_I_6 sampling site near the North Sea island Helgoland.

The tree was inferred by using the Maximum Likelihood method with the JTT matrix-based model. The tree with the highest log likelihood (-42104.84) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. There were a total of 1,206 positions in the final dataset. Evolutionary analyses were conducted in MEGA X. The bootstrap value is 500.

Fig. 4. ShGH76 is the only key endo-acting enzyme in the Hel_I_6 α-mannan PUL.

A α-Mannan PUL of Salegentibacter sp. Hel_I_6. The asterisk marks remnants of a transposase gene. B 3D structure-guided multiple sequence alignment of the Salegentibacter sp. Hel_I_6 GH76 (ShGH76) with homologs. The conserved two catalytic aspartate residues (DD) are highlighted by a red box. LiGH76: Listeria innocua GH76; BcGH76: Bacillus circulans GH76; BTGH76-MD40, BT3782_GH76, BT3792_GH76 and BT2949_GH76: Bacteroides thetaiotaomicron GH76s.

Fig. 5. ShGH76 exhibits endo-mannanase activity on linear yeast α-mannan and linear mannooligosaccharides.

A Activity of ShGH76^WT on yeast α-mannan from pH 3.0-11.0 using a PAHBAH assay; error bars represent S.D. over at least three replicates. B HPAEC-PAD chromatograms of linear yeast α-mannan (LYM) incubated with ShGH76^WT for 30 min and after 16 h. The heat-killed ShGH76^WT is control. The peaks were labeled with corresponding products formed. C Affinity gel electrophoresis suggesting yeast α-mannan binding by the ShGH76^Ala mutant (arrow). D HPAEC-PAD chromatograms of mannoheptose (Man7) in the presence of the ShGH76^WT. Peaks are labeled with corresponding products. Man1: mannose, Man2: α-1,6-linked-mannobiose, Man3: α-1,6-linked-mannotriose, Man4: α-1,6-linked-mannotetrose, Man5: α-1,6-linked-mannopentose, Man6: α-1,6-linked-mannohexose, and Man7: α-1,6-linked-mannoheptose.

Fig. 6. X-ray crystal structures of ShGH76 variants indicated classical single-domain (α/α)6-barrel fold.

A X-ray crystal structure of ShGH76^WT displayed in cartoon format with a rainbow color scheme (NT = blue; CT = red) showing single-domain (α/α)₆ fold viewed along the barrel axis with the active site in the center. B Overview of the ShGH76^Ala mutant structure in complex with mannotetrose (Man4: yellow stick) in cartoon format with a gray color showing single-domain (α/α)₆ fold viewed along barrel axis having active site in the center. MES and glycerol are also indicated. C 3D-crystal structure of the ShGH76^Asn mutant in complex with mannotriose (Man3: yellow stick) displayed in cartoon format with gray color showing single-domain (α/α)₆ fold viewed along the barrel axis with active site in the center. Three Ca²⁺ ions are shown as magenta spheres.

Fig. 7. Highly conserved and negatively charged ShGH76 active site remains intact upon ligand binding.

A Structural superimposition of ShGH76^WT (gray) and BT3792 (green, PDB ID: 4C1S) showing major variations in secondary structures highlighted in red color. B Electrostatic surface potential representation of ShGH76^WT showing the predominantly negatively charged active site (red: negatively charged; blue: positively charged). C Surface view of ShGH76^WT showing the highly conserved (magenta) active site pocket. Color intensity indicates conservation strength. The black box indicates the active site.

Fig. 8. Ligand adopted a kinked conformation in the ShGH76 active site.

A Superimposition of active site forming residues of all three ShGH76 structures, ShGH76^WT (gray), ShGH76^Ala (green) and ShGH76^Asn (wheat). B Surface-view representation of ShGH76^WT showing the ligand pocket and catalytic residues (red). The aromatic residues lining the active site are shown in cyan. The modeled Man3 (yellow) and Man4 (blue) are shown in stick representation.