Figure 4.

¹H NMR spectroscopy reveals the mode of action of EpCgS2. (a) κ-carrageenan incubated with BovGH16, yielding oligo-κ-neocarrageenans. (b) ι-carrageenan incubated with BovGH16, yielding oligo-ι-neocarrageenans. (c) ι-carrageenan coincubated with BovGH16 and EpCgS2. Desulfation of oligo-ι-neocarrageenans by EpCgS2 leads to the formation of κ-carrageenan moieties at the nonreducing end, as indicated by the emerging signals for the α-anomeric proton of DAnr-H1 at 5.08 ppm in the ¹H NMR spectrum. (d) ι-carrageenan coincubated with BovGH16 and CaCgS1. Desulfation of oligo-ι-neocarrageenan by CaCgS1 leads to the formation of α-neocarrageenan moieties, as indicated by the emerging signal for the α-anomeric proton of DA2Snr-H1 at 5.20 ppm in the ¹H NMR spectrum. (e) ι-carrageenan sequentially incubated with BovGH16, CaCgS1 and EpCgS2. The prior incubation of the BovGH16 generated oligo-ι-neocarrageenans with CaCgS1 leads to the formation of α-neocarrageenan oligosaccharides (5.20 ppm). Subsequent incubation of the reaction product with EpCgS2 further desulfates α-neocarrageenan to β-neocarrageenan oligosaccharides, indicated by the α-anomeric signal DAnr-H1 (β) at around 5.06 ppm. The structures next to the spectra display the respective, sequential sulfatase reactions. The reactions were performed at 37 °C and terminated by heat-inactivation of the enzyme. Enzymes were removed by centrifugation, and the supernatant was dried before resuspension in D₂O. ¹H NMR spectra were recorded at 70 °C.