The Cyclization of the 3,6-Anhydro-Galactose Ring of ι-Carrageenan Is Catalyzed by Two d-Galactose-2,6-Sulfurylases in the Red Alga Chondrus crispus

Abstract

Carrageenans are sulfated galactans found in the cell walls of numerous red seaweeds (Rhodophyta). They are classified according to the number and the position of sulfate ester groups and the occurrence of 3,6-anhydro-galactose. Although the carrageenan biosynthesis pathway is not fully understood, it is usually accepted that the last step consists of the formation of a 3,6-anhydro ring found in k-and i-carrageenans through the enzymatic conversion of D-galactose-6-sulfate or D-galactose-2,6-disulfate occurring in m-and n-carrageenan, respectively. We purified two enzymes, sulfurylase I (65 kD) and sulfurylase II (32 kD), that are able to catalyze the conversion of n-into i-carrageenan. We compared their sulfate release rates (i.e. arising from the formation of the anhydro ring) with the viscosity of the solution and demonstrated two distinct modes of action. In addition, we found that some mixtures of sulfurylase I and II lead to the formation of carrageenan solutions with unexpectedly low viscosities. We discuss the implication of these findings for the assembly of a densely aggregated matrix in red algal cell walls.Agars and carrageenans are the most abundant components of the cell walls in numerous red algae (Rhodophyta) and can represent up to 50% of algal dry weight. These sulfated galactans are densely packed in the cell wall in a three-dimensional solid network of pseudocrystalline fibers, which assemble during the deposition of cell wall macromolecules (Craigie, 1990).

Marine Polysaccharide Sulfatases

“…incubated on porphyran purified from the same algae. Similar approaches conducted with carrageenophyte algae also lead to the formation of an anhydro-ring in κ-and ι-carrageenan (Wong and Craigie, 1978;Zinoun et al, 1997;Genicot-Joncour et al, 2009). The pure enzyme was sequenced and expression of the corresponding gene was attempted in E. coli without success.…”

Biochemical and structural investigation of two paralogous glycoside hydrolases fromZobellia galactanivorans: novel insights into the evolution, dimerization plasticity and catalytic mechanism of the GH117 family

“…The formation of 3,6anhydrogalactose moieties is catalyzed at the polymer level by galactose-6-sulfurylases. These unique enzymes convert galactose 6-sulfate (L6S or D6S monomer) into 3,6-anhydrogalactose, releasing a free sulfate ion (Lawson & Rees, 1970;Genicot-Joncour et al, 2009). However, knowledge of the biosynthesis of sulfated galactans remains very limited and the first global view of the carbohydrate metabolism of red seaweeds has only recently been provided by the genome sequencing of the carrageenophyte Chondrus crispus (Collé n et al, 2013(Collé n et al, , 2014.…”

Towards a Synthetic Strategy for the Ten Canonical Carrageenan Oligosaccharides – Synthesis of a Protected γ‐Carrageenan Tetrasaccharide

“…It has been shown that hot alkaline treatment of the D6S carrageenans (namely carrageenan having a 4‐linked‐α‐ d ‐galactopyranose residue sulfated at the 6 position) leads to cyclization to form the 3,6‐anhydro ring accompanied by a ring flip from 4 C 1 to 1 C 4 (see Figure a) , . In algae this reaction is catalyzed by enzymes such as sulfohydrolases, or galactose‐6‐sulfurylases, which have been isolated from algal cells. The activity of sulfurylases I and II catalyzing the conversion of ν‐carrageenan to ι‐carrageenan was demonstrated in vitro and proved to be specific to this type of carrageenan as it was inactive on µ‐carrageenan …”

“…In algae this reaction is catalyzed by enzymes such as sulfohydrolases, or galactose‐6‐sulfurylases, which have been isolated from algal cells. The activity of sulfurylases I and II catalyzing the conversion of ν‐carrageenan to ι‐carrageenan was demonstrated in vitro and proved to be specific to this type of carrageenan as it was inactive on µ‐carrageenan …”

“…The activity of sulfurylases I and II catalyzing the conversion of ν-carrageenan to ι-carrageenan was demonstrated in vitro and proved to be specific to this type of carrageenan as it was inactive on μ-carrageenan. [26] The chemical synthesis of oligocarrageenans requires the formation of a backbone of alternating 3-linked--D-galactopyran-ose and 4-linked-α-D-galactopyranose units, followed by selective sulfation of various positions depending on the targeted carrageenan. The strategy we investigated is shown in Figure 2 and uses a block-wise assembly.…”