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

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.


Supplementary Figure 3. Detection of bacterial cell extracts producing X-SO4 specific sulfatases.
Cells were grown on agar plates containing X-SO4, which when cleaved produces blue coloring. Cell extracts were added to exercised holes in the plate. The MB87 and MB104 strains showed X-SO4 specific sulfatase activity, while the MB47 strain showed comparably higher X-SO4 specific sulfatase activity.
Negative control strain: MB04.    The pyrroloquinoline quinone-dependent oxidoreductase activity was demonstrated for the CC1G_09525 protein of Coprinopsis cinerea.

CBM5 4
Modules of approx. 60 residues found in bacterial enzymes. Chitin-binding described in several cases. Distantly related to the CBM12 family.

CBM9 3
Modules of approx. 170 residues found so far only in xylanases. The cellulose-binding function has been demonstrated in one case.

CBM13 3
Modules of approx. 150 residues which always appear as a threefold internal repeat. The only apparent exception to this, xylanase II of Actinomadura sp. FC7 (GenBank U08894), is in fact not completely sequenced. These modules were first identified in several plant lectins such as ricin or agglutinin of Ricinus communis which bind galactose residues. The three-dimensional structure of a plant lectin has been determined and displays a pseudo-threefold symmetry in accord with the observed sequence threefold repeat. These modules have since been found in a number of other proteins of various functions including glycoside hydrolases and glycosyltransferases. While in the plant lectins this module binds mannose, binding to xylan has been demonstrated in the Streptomyces lividans xylanase A and arabinofuranosidase B. Binding to GalNAc has been shown for the corresponding module of GalNAc transferase 4. For the other proteins, the binding specificity of these modules has not been established. The pseudo three-fold symmetry of the CBM13 module has now been confirmed in the 3-D structure of the intact, two-domain, xylanase of Streptomyces olivaceoviridis. CBM16 1 Carbohydrate-binding module 16. Binding to cellulose and glucomannan CBM32 2 Binding to galactose and lactose has been demonstrated for the module of Micromonospora viridifaciens sialidase (PMID: 16239725). Binding to polygalacturonic acid has been shown for a Yersinia member (PMID: 17292916). Binding to LacNAc (beta-D-galactosyl-1,4-beta-D-Nacetylglucosamine) has been shown for an N-acetylglucosaminidase from Clostridium perfingens (PMID: 16990278).

CBM35 1
Modules of approx. 130 residues. A module that is conserved in three Cellvibrio xylan-degrading enzymes binds to xylan and the interaction is calcium dependent, while a module from a Cellvibrio mannanase binds to decorated soluble mannans and mannooligosaccharides. A module in a CBM40 1 Modules of approx. 200 residues, found at the N-terminus of GH33 sialidases. Can also be found inserted in the beta-propeller of GH33 sialidases. The sialic acid binding function has been demonstrated for the N-terminal CBM40 of Vibrio cholerae sialidase CBM41 1 Modules of approx. 100 residues found in primarily in bacterial pullulanases. The N-terminal module from Thermotoga maritima Pul13 has been shown to bind to the alpha-glucans amylose, amylopectin, pullulan, and oligosaccharide fragments derived from these polysaccharides CBM44 1 The C-terminal CBM44 module of the Clostridium thermocellum enzyme has been demonstrated to bind equally well cellulose and xyloglucan CBM48 4 Modules of approx. 100 residues with glycogen-binding function, appended to GH13 modules. Also found in the beta subunit (glycogen-binding) of AMP-activated protein kinases (AMPK) CBM50 8 Modules of approx. 50 residues found attached to various enzymes from families GH18, GH19, GH23, GH24, GH25 and GH73, i.e. enzymes cleaving either chitin or peptidoglycan. Binding to chitopentaose demonstrated in the case of Pteris ryukyuensis chitinase A. CBM50 modules are also found in a multitude of other enzymes targeting the petidoglycan such as peptidases and amidases. These enzymes are not reported in the list below.

CBM66 1
The CBM66 module, derived from the Bacillus subtilis exo-acting beta-fructosidase SacC, targets the terminal fructoside residue of fructans. CBM69 1 Starch-binding function demonstrated in one case; distantly related to families CBM20 and CBM48