Sulfohydrolase Activity and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)

Abstract

An enzyme catalyzing the conversion of i-to X-carrageenan Cell walls of red seaweeds frequently contain conspicuous amounts of galactans among which the carrageenans are a commercially important group. These polysaccharides are composed of regularly alternating a-1,3; 1-1,4-linked D-galactose residues which differ in degree and position of sulfate esterification, and in their content of 3,6-anhydrogalactose (12). As early as 1963 Rees (1 1) postulated that a "A-like" fraction from Chondrus crispus may be the biological precursor of K-carrageenan in a manner analogous to that seen in the biosynthesis of porphyran. In the latter case L-galactose-6-sulfate residues in the polymer were converted to 3,6-anhydrogalactose by an enzyme present in Porphyra umbilicalis (10). The predicted enzyme activity was detected in extracts of Gigartina stellata (6), but similar preparations from C. crispus appeared to be inactive (Rees, personal communication).The present paper is the first characterization of an enzyme from C. crispus which eliminates sulfate from p-carrageenan to form the 3,6-anhydrogalactose of K-carrageenan. The enzyme was not sufficiently characterized to be listed in the 1972 Enzyme Nomenclature. Because the mechanism ofthe reaction is unknown the name "sulfohydrolase" used in the present report is based on the release of sulfate from the polymer. Enzyme Extraction from Haploid Plants. Apical 2-to 3-cm pieces of C. crispus (100 g) were frozen in liquid N2 and pulverized in a ball mill for 2 min. The resulting flour was allowed to thaw and 150 ml of cold buffer containing 0.05 M Tris-HCl, 0.5 M KCI, and 0.01 M 2-mercaptoethanol (pH 9.5) were added. The suspension was stirred for 2 hr and left overnight in crushed ice. The crude homogenate was recovered after centrifugation at 15,000g for 20 min. Cold saturated (NH4)2SO4 was added and the fraction precipitating between 2.65 and 4.24 M was collected by centrifugation. The precipitate, containing some polysaccharides as well as the active protein, was dissolved in 25 ml of 0.05 M Tris-HCl with 2-mercaptoethanol (pH 7.1) and dialyzed overnight in the same buffer. This enzyme preparation was routinely used. MATERIALSPurification of DEAE-CeUlulose. The protein (150 mg) from the (NH4)2SO4 precipitation was applied to a column (25 x 3 cm) of DEAE-cellulose previously equilibrated with 0.05 M borate containing 0.01 M 2-mercaptoethanol (pH 7.5). The column was washed with 250 ml of the same buffer followed with I M KCl.Three-ml fractions were collected every 1.6 min. Fractions 141 to 159 containing the active enzyme were pooled and treated with saturated (NH4)S04 until 4.24 M was reached. The precipitate collected by centrifugation was redissolved in 0.05 M Tris-HCl containing 0.01 M 2-mercaptoethanol (pH 7.1) and dialyzed overnight in the same buffer.Preparation

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

“…This reaction was also shown to occur in vivo (Hemmingson et al, 1996a(Hemmingson et al, , 1996b. The formation of the k-and i-carrageenan anhydro rings was also observed after incubation of m-and n-carrageenans, respectively, with red algal protein extracts that have been named sulfohydrolases (Wong and Craigie, 1978;Zinoun et al, 1997). These observations confirm that the formation of anhydro rings is catalyzed at the final step of biosynthesis.…”

“…The pH optima were also similar and measured at about pH 8 to 9 and pH 7 to 8 for sulfurylase I and II, respectively. Wong and Craigie (1978) reported a pH optimum of 6.5 for the C. crispus sulfohydrolase that converts m-into k-carrageenan. Biochemical parameters-apparent K m , V max , and k cat of the enzymes reported in Table II-were also very similar between the two enzymes.…”

“…However, based on the structures of agarobiose and carrabiose moieties that co-occur in polysaccharide chains, the proposed sequences of catalytic events accounting for the chemical structures of agars and carrageenans usually assume that three classes of enzymes are involved: galactosyl transferases, sulfotransferases, and Gal-6-sulfurylases (in agarose; Rees, 1961aRees, , 1961b, referred to as "sulfohydrolases" in carrageenans (Wong and Craigie, 1978). The a-and b-galactosyl transferases catalyze the polymerization of the Gal backbone.…”

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.…”

Glycosulphatase from Pseudomonas carrageenovora. Purification and Some Properties

“…Sulphatases have been detected ubiquitously in organisms [l -31 pertinent examples being bacteria [4,5] and algae [6,7]. In the degradation of chondroitin sulphate by Proteus vulgaris [4] only disaccharide oligomers are susceptible to the relevant sulphatases.…”