The cyclization of the 3,6-anhydro-galactose ring of iota-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 kappa- and iota-carrageenans through the enzymatic conversion of d-galactose-6-sulfate or d-galactose-2,6-disulfate occurring in mu- and nu-carrageenan, respectively. We purified two enzymes, sulfurylase I (65 kD) and sulfurylase II (32 kD), that are able to catalyze the conversion of nu- into iota-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.

Figure 1.

Formation of agarose, κ-, and ι-carrageenan repeating disaccharide moieties generated enzymatically from their respective biosynthetic precursors, porphyran, μ-, and ν-carrageenan.

Figure 2.

Chromatographic purification of sulfurylases I and II on a DEAE-Sepharose fast-flow column (2 × 22 cm). Elution of proteins was performed using an increasing gradient of NaCl. The concentration of released sulfate (white circles) and the viscosity (black squares) of the resulting polymer obtained after incubation with ν-/ι-carrageenan are presented for the different fractions.

Figure 3.

SDS-PAGE of purified sulfurylase I (lane A) and II (lane B) stained with Coomassie Brilliant Blue R-250. Lanes M, Size markers.

Figure 4.

¹H NMR spectra of the conversion kinetics of ν- to ι-carrabiose using ν-/ι-carrageenan (A) and hybrid ν-/ι-/κ-/μ-carrageenan (B) as substrates. These substrates were incubated with sulfurylase I (52 ng) at 40°C for up to 24 h. The anomeric protons of the various carrabiose units (ν-, D2S,6S-H1; ι-, DA2S-H1; μ-, D6S-H1; κ-, DA-H1) as well as the protons corresponding to ν-carrabiose moieties are indicated on the spectra.

Figure 5.

Variation in the viscosity of the incubation medium determined after treatment of ι-/ν-carrageenan with sulfurylase I and II as a function of the concentration of released sulfate. Error bars correspond to the sd determined from three independent experiments.

Figure 6.

Viscosity (white squares) and concentration of released sulfate (black triangles) after 12 h of incubation of ι-/ν-carrageenan with increasing sulfurylase I (Sulf I)-to-sulfurylase II (Sulf II) ratios. Error bars correspond to the sd determined from three independent experiments.