A novel thermostable prokaryotic fucoidan active sulfatase PsFucS1 with an unusual quaternary hexameric structure

Abstract

Fucoidans are sulfated, fucose-rich marine polysaccharides primarily found in cell walls of brown seaweeds (macroalgae). Fucoidans are known to possess beneficial bioactivities depending on their structure and sulfation degree. Here, we report the first functional characterization and the first crystal structure of a prokaryotic sulfatase, PsFucS1, belonging to sulfatase subfamily S1_13, able to release sulfate from fucoidan oligosaccharides. PsFucS1 was identified in the genome of a Pseudoalteromonas sp. isolated from sea cucumber gut. PsFucS1 (57 kDa) is Ca2+ dependent and has an unusually high optimal temperature (68 °C) and thermostability. Further, the PsFucS1 displays a unique quaternary hexameric structure comprising a tight trimeric dimer complex. The structural data imply that this hexamer formation results from an uncommon interaction of each PsFucS1 monomer that is oriented perpendicular to the common dimer interface (~ 1500 Å2) that can be found in analogous sulfatases. The uncommon interaction involves interfacing (1246 Å2) through a bundle of α-helices in the N-terminal domain to form a trimeric ring structure. The high thermostability may be related to this unusual quaternary hexameric structure formation that is suggested to represent a novel protein thermostabilization mechanism.

Herbivorous fish microbiome adaptations to sulfated dietary polysaccharides

“…and in host-associated gut microbiota from humans [37], sea urchins [38], sea cucumbers [39], marine snails [23], and surgeon shes [40]. Some bacteria may use related enzyme functions to break down sulfated glycosaminoglycans from eukaryotic host tissue [41,42].…”

Sulfatases: Critical Enzymes for Algal Polysaccharide Processing

“…Considerable effort has recently gone into trying to understand the depolymerisation of sulphate containing algal polysaccharides. Of the recently characterised marine carbohydrate-specific sulfatases, only seven with confirmed biological activity have been structurally characterised and four of these have been determined with substrate bound in the active site ( Hettle et al, 2018 , 2019 ; Silchenko et al, 2018 ; Mikkelsen et al, 2021 ; Robb et al, 2022 ).…”

“… Marine carbohydrate-specific sulfatases in complex with their biological substrates. The X-ray crystal structure of PfS1_19A C78S in complex with a κ-ι-κ-carrageenan hexasaccharide [PDB ID: 6B0J, Hettle et al (2018) ] (A) , PfS1_19B C77S in complex with a κ-carrageenan disaccharide [PDB ID: 6PSM, Hettle et al (2019) ] (B) , PfS1_NC C84S in complex with an ι-carrageenan disaccharide [PDB ID: 6PT6, Hettle et al (2019) ] (C) , BuS1_11 S80 in complex with neoporphyrabiose [PDB ID: 7LJ2, Robb et al (2022) ] (D) , P18_S1_7 [PDB ID: 6HHM, Reisky et al (2019) ] (E) , P36_S1_25 [PDB ID: 6HR5, Reisky et al (2019) ] (F) and PsFuc1 [PDB ID: 7AJ0, Mikkelsen et al (2021) ] (G) . In all panels, S-subsite residues are shown in pink sticks, integer-subsites as purple sticks, the calcium ion is represented as a yellow sphere, and the subsites are labelled in red.…”

“…MB47. Through amino acid sequence comparisons using the SulfAtlas Database, of these putative sulfatases, PsSUL1, 3, 4 and PsFucS1 belong to family S1_13 while PsSUL2 belongs to S1_4 ( Mikkelsen et al, 2021 ). PsFucS1 is the only one of these enzymes to have demonstrated sulfatase activity on fucoidan and its x-ray crystal structure has been solved revealing the canonical S1 family sulfatase fold including the S-subsite found in the active site pocket ( Figure 4G ; Mikkelsen et al, 2021 ).…”

“… X-ray crystal structures of marine carbohydrate-specific sulfatases highlighting the importance of the C-terminal sub-domain. The X-ray crystal structure showing the solvent accessible surface of PfS1_NC C84S in complex with an ι-carrageenan disaccharide [PDB ID: 6PT6, Hettle et al (2019) ] (A) , PfS1_19BC77S in complex with a κ-carrageenan disaccharide [PDB ID: 6PSM, Hettle et al (2019) ] (B) , BuS1_11 S80 in complex with neoporphyrabiose [PDB ID: 7LJ2, Robb et al (2022) ] (C) , P36_S1_25 [PDB ID: 6HR5, Reisky et al (2019) ] (D) , PsFuc1 [PDB ID: 7AJ0, Mikkelsen et al (2021) ] (E) , PsS1_19A C78S in complex with a κ-ι-κ-carrageenan hexasaccharide [PDB ID: 6B0J, Hettle et al (2018) ] (F) and P18_S1_7 [PDB ID: 6HHM, Reisky et al (2019) ] (G) . In all panels, the N-terminal sub-domain is coloured white, the C-terminal sub-domain is coloured ruby.…”