Structural insights into marine carbohydrate degradation by family GH16 κ-carrageenases

Abstract

Carrageenans are sulfated α-1,3-β-1,4-galactans found in the cell wall of some red algae that are practically valuable for their gelation and biomimetic properties but also serve as a potential carbon source for marine bacteria. Carbohydrate degradation has been studied extensively for terrestrial plant/bacterial systems, but sulfation is not present in these cases, meaning the marine enzymes used to degrade carrageenans must possess unique features to recognize these modifications. To gain insights into these features, we have focused on κ-carrageenases from two distant bacterial phyla, which belong to glycoside hydrolase family 16 and cleave the β-1,4 linkage of κ-carrageenan. We have solved the crystal structure of the catalytic module of ZgCgkA from Zobellia galactanivorans at 1.66 Å resolution and compared it with the only other structure available, that of PcCgkA from Pseudoalteromonas carrageenovora 9^T (ATCC 43555^T). We also describe the first substrate complex in the inactivated mutant form of PcCgkA at 1.7 Å resolution. The structural and biochemical comparison of these enzymes suggests key determinants that underlie the functional properties of this subfamily. In particular, we identified several arginine residues that interact with the polyanionic substrate, and confirmed the functional relevance of these amino acids using a targeted mutagenesis strategy. These results give new insight into the diversity of the κ-carrageenase subfamily. The phylogenetic analyses show the presence of several distinct clades of enzymes that relate to differences in modes of action or subtle differences within the same substrate specificity, matching the hybrid character of the κ-carrageenan polymer.

Keywords: carbohydrate recognition; crystal structure; enzyme kinetics; enzyme-substrate complex; kappa-carrageenan; polysaccharide; processivity; site-directed mutagenesis; sulfated polysaccharides.

Figure 1.

Schematic representations of the chemical structure of κ-neocarratetraose. At the top, the chemical representations of the units show the standard chair conformation. At the bottom, the simplified scheme follows the same order of monosaccharides and glycosidic linkages as above. NR, non-reducing end; R, reducing end.

Figure 2.

Comparison of the catalytic modules of PcCgkA_GH16 and ZgCgkA_GH16. A, multiple-sequence alignment of the catalytic modules of both enzymes. The alignment was produced with MultAlin, and the figure was produced using ESPript version 3.x software. Secondary structure elements are symbolized with arrows for β-sheets, curls for α-helix, and T for turns; the “finger” regions surrounding the catalytic channel are boxed and numbered from F1 to F6; the sequence divergences that have structural consequences are boxed in green; the numbers 1 in green refer to the cysteine residues involved in the single disulfide bond of PcCgkA_GH16; the black stars above the sequence highlight the amino acids that were mutated into alanine for PcCgkA_GH16 in this study. B, schematic representation of the structural fold of ZgCgkA_GH16. The color code is graduated from blue (N terminus) to red (C terminus) along the polypeptide chain. Catalytic residues Glu¹⁵⁹-Asp¹⁶¹-Glu¹⁶⁴ are shown as sticks and colored in red. C, schematic representation of the structural fold of PcCgkA_GH16-E168D in complex with a κ-neocarratetraose, shown in stick representation (carbon atoms are white, oxygen atoms are red, and sulfur atoms are orange). The color code of the polypeptide chain is the same as in B. Catalytic residues Glu¹⁶³-Asp¹⁶⁵-E168D are shown as sticks and colored in red.

Figure 3.

Phylogenetic tree of the currently sequenced κ-carrageenases. The multiple alignments were done with 37 sequences of catalytic modules of κ-carrageenases. Clades A–D are defined according to the bootstrap values. Structural common features are identified for these clades and are represented in the circles. Purple, presence of finger F2 and absence of finger F4; blue, presence of finger F4 and absence of finger F2; black, presence of both fingers F2 and F4, subgroup I being the closer in sequence similarity for F2 and F4 motifs and subgroup II showing the higher divergence. Blue arrows identify the six κ-carrageenases from Algibacter sp. SK-16.

Figure 4.

Kinetics of degradation of κ-carrageenan. A and B, reducing sugar analysis in solution (A) and microgels (B) of κ-carrageenan for PcCgkA_GH16 (white circles), ZgCgkA_GH16 (light gray squares), and ZgCgkA_{GH16-CBM16-PorSS} (dark gray triangles). Error bars, S.D. of triplicate determinations. C and D, HPAEC analyses of degradation products of ZgCgkA_GH16 in solution (C) and in microgel (D). E and F, HPAEC analyses of degradation products of ZgCgkA_{GH16-CBM16-PorSS} in solution (E) and in microgel (F). For C, D, E, and F, different curves correspond to kinetics at t₀ = 0, t₁ = 15 min, t₂ = 45 min, t₃ = 24 h, t₄ = 1 week. The asterisks in C–F designate peaks corresponding to noncharacterized reaction products.

Figure 5.

Structural representations of the substrate–enzyme interactions in PcCgkA_GH16-E168D. A, Fourier difference map (F_o − F_c) displaying the electron density (blue mesh) in the active site at a level of 3.5 σ that indicates the presence of the DP4 oligosaccharide (stick representation with the same color code as in Fig. 2). The catalytic sub-binding sites are numbered from −4 to −1. The polypeptide chain is represented as a purple schematic. B, schematic representation of the enzyme-substrate complex (produced with ChemDraw). All residues that interact with the substrate molecule are displayed. Possible hydrogen bonds are represented in dashes, and those established with sulfate groups are underlined in yellow, as well as the labels of the corresponding residues; water molecules are drawn as spheres; hydrophobic interactions are symbolized with arcs; the mutation E168D is preceded by an asterisk; the three arginine residues that were mutated into alanines are identified by a star; the three catalytic residues are labeled in red, and red waves symbolize the hydrogen bonds that are subsequently formed during the catalytic cleavage of the glycosidic linkage. C, schematic representation of PcCgkA_GH16-E168D, showing the positions of the amino acids mutated into alanine, with respect to the catalytic sub-binding sites. The positions of residues Trp²⁶⁶ and Gln¹⁷¹ are on either side of the subsite +1. D, superposition of PcCgkA_GH16 (PDB code 1DYP) in gray and PcCgkA_GH16-E168D in purple, in complex with a neocarratetraose in light gray (DA-G4S-DA-G4S) (PDB code 5OCQ).

Figure 6.

Structural comparison of the catalytic modules of PcCgkA_GH16-E168D and ZgCgkA_GH16 and their interactions with κ-neocarratetraose. A, schematic representation of the overall structure with the six identified fingers numbered from F1 to F6; the catalytic triad is identified with red letters; the substrate is symbolized by the orange arrow oriented from its non-reducing (NR) to its reducing (R) end; the specific fingers F2 and F4 are identified with a purple and a blue ring, respectively. B, ribbon representation of the two superposed structures with the oligosaccharide in orange, the catalytic triad in red, and the specific structural features of both enzymes in purple for PcCgkA_GH16-E168D and blue for ZgCgkA_GH16. C, ZgCgkA_GH16 surface representation with its specific structural features in blue (top) superimposed with the oligosaccharide trapped in PcCgkA_GH16-E168D in orange and PcCgkA_GH16-E168D surface representation with its specific structural features in purple (bottom) and the oligosaccharide in orange. The tilt of the positive substrate binding sites by 45° is highlighted in orange. (+) and (−) indicate the regions of the respective subsites. D, schematic representation of the interactions between the catalytic subsites of ZgCgkA_GH16 (blue) and PcCgkA_GH16-E168D (purple) with a DP8 κ-carrageenan; the red dashed arrow symbolizes the cleavage site; white arrows symbolize the proposed direction of enzymatic processivity.