The agar-specific hydrolase Zg AgaC from the marine bacterium Zobellia galactanivorans defines a new GH16 protein subfamily

Abstract

Agars are sulfated galactans from red macroalgae and are composed of a d-galactose (G unit) and l-galactose (L unit) alternatively linked by α-1,3 and β-1,4 glycosidic bonds. These polysaccharides display high complexity, with numerous modifications of their backbone (e.g. presence of a 3,6-anhydro-bridge (LA unit) and sulfations and methylation). Currently, bacterial polysaccharidases that hydrolyze agars (β-agarases and β-porphyranases) have been characterized on simple agarose and more rarely on porphyran, a polymer containing both agarobiose (G-LA) and porphyranobiose (GL6S) motifs. How bacteria can degrade complex agars remains therefore an open question. Here, we studied an enzyme from the marine bacterium Zobellia galactanivorans (ZgAgaC) that is distantly related to the glycoside hydrolase 16 (GH16) family β-agarases and β-porphyranases. Using a large red algae collection, we demonstrate that ZgAgaC hydrolyzes not only agarose but also complex agars from Ceramiales species. Using tandem MS analysis, we elucidated the structure of a purified hexasaccharide product, L6S-G-LA2Me-G(2Pentose)-LA2S-G, released by the activity of ZgAgaC on agar extracted from Osmundea pinnatifida By resolving the crystal structure of ZgAgaC at high resolution (1.3 Å) and comparison with the structures of ZgAgaB and ZgPorA in complex with their respective substrates, we determined that ZgAgaC recognizes agarose via a mechanism different from that of classical β-agarases. Moreover, we identified conserved residues involved in the binding of complex oligoagars and demonstrate a probable influence of the acidic polysaccharide's pH microenvironment on hydrolase activity. Finally, a phylogenetic analysis supported the notion that ZgAgaC homologs define a new GH16 subfamily distinct from β-porphyranases and classical β-agarases.

Keywords: GH16; agar; algae; bacteria; crystal structure; evolution; glycoside hydrolase; marine bacteria; mass spectrometry; pH microenvironment; polysaccharide; red algae; sulfated polysaccharide.

Figure 1.

A, schematic of the primary structure of the agaC-encoded protein. The native protein comprises a lipoprotein signal peptide (yellow box), a low-complexity region rich in glutamate and lysine (green box), and a GH16 family catalytic module (blue box). B, schematic of the sequence of the recombinant ZgAgaC protein. ZgAgaC includes a noncleavable N-terminal His tag (purple box) and the GH16 family catalytic module (blue box). C, schematic of the sequence of the GST-ZgAgaC_E193T fusion protein. This recombinant protein includes an N-terminal GST domain (orange box), a linker containing the cleavage site for the H3C protease (red box), and the GH16 family catalytic module (blue box). In the three schematics, the size and the limits of the domains are indicated. D, structure-based sequence alignment of ZgAgaC with characterized β-agarases and β-porphyranases. α-Helices and β-strands are represented as helices and arrows, respectively, and β-turns are marked with TT. This sequence alignment was created using the following sequences from the Protein Data Bank: the β-agarases ZgAgaA (1O4Y) and ZgAgaB (1O4Z) and the β-porphyranases ZgPorA (3ILF) and ZgPorB (3JUU). All of these GH16 enzymes originate from Z. galactanivorans. Dark shaded boxes enclose invariant positions, and light shaded boxes show positions with similar residues. The green 1 numbers correspond to a cysteine pair involved in a disulfide bridge in ZgAgaC. The figure was created with ESPript 3.0 (71).

Figure 2.

Phylogenetic tree of the galactanases of the GH16 family. The phylogenetic tree was generated using the maximum likelihood approach with the program MEGA version 6 (57). Bootstrap numbers are indicated. The tree was rooted by the laminarinases ZgLamA and ZgLamB from Z. galactanivorans. The clades of the κ-carrageenases, the ZgPorA homologues, the ZgPorB homologues, and the classical β-agarases have been collapsed. The sequence of ZgAgaC from Z. galactanivorans is marked with a black diamond.

Figure 3.

FACE of the oligosaccharides released by the action of ZgPorB, ZgAgaB, and ZgAgaC on the red algae P. elongata (A) and O. pinnatifida (B). The oligosaccharide products were coupled with the negatively charged ANTS prior to FACE analysis. The direction of migration is indicated by a yellow arrow. The oligosaccharide bands, which are specifically produced by ZgAgaC are shown by green arrows. The oligosaccharide bands, which are lacking in the ZgAgaC lane compared with the ZgAgaB and ZgPorB lanes are shown by red arrows. The bands in the control lanes are either unbound ANTS or oligosaccharides naturally present in the algal samples and released by the grinding process.

Figure 4.

Hydrolysis of agarose by ZgAgaC monitored by FACE. The reaction products were labeled with the ANTS fluorophore and migrated onto a 31% polyacrylamide gel. The time points are shown in minutes.

Figure 5.

pH dependence of ZgAgaC. The pH dependence of ZgAgaC was determined for the neutral polysaccharide agarose (0.4% (w/v), blue dot) and for the sulfated agar from O. pinnatifida (0.4% (w/v), orange dot). The rates of reactions are expressed in s⁻¹ (k_obs). Error bars, S.D.

Figure 6.

ESI-MS measurements of the three fractions (OP44 (A), OP36 (B), and OP30 (C)) that contained the smallest oligosaccharides identified by FACE gels. Annotations were deduced from the exact mass measurements. The black star indicates noncommon species with one 3,6-anhydro-l-galactose replaced by a l-galactose in the regular moieties.

Figure 7.

XUV-DPI tandem MS spectrum of the doubly charged species isolated at m/z 629.1 as a [M − 2H]²⁻ ion. The spectrum was recorded following 18-eV photon irradiation for 500 ms. With the aim of better readability, the mass range was split into three parts. Red, fully sulfated fragments. Blue, fragments with one sulfate loss. Triangle, water losses. Signal was accumulated over 2 min.

Figure 8.

Interaction of ZgAgaC with the His tag of a symmetrical ZgAgaC chain. A, global view of ZgAgaC (colored in purple) interacting with its symmetrical molecule (colored in magenta). The two protein chains are shown in cartoon representation, except for the His tag of a symmetrical molecule, which is shown in stick representation. B, a view zooming in on the −1 subsite of ZgAgaC (colored in purple) with a bound glycerol molecule (colored in orange) hydrogen-bonded to Trp²⁹¹ and His⁶³ of the symmetrical His tag (colored in magenta). C, a view zooming in on the interaction between Trp¹¹⁰ (colored in purple) and His⁶¹ of the symmetrical His tag (colored in magenta).

Figure 9.

Fold and topology of ZgAgaC. A, ZgAgaC adopts a β-jelly-roll fold (cartoon representation). The nucleophile Glu¹⁸⁸ and the acid/base catalyst Glu¹⁹³ are shown as sticks. The gray and red spheres represent a bound magnesium ion and two coordinating water molecules, respectively. B, close-up view of the Mg²⁺-binding site. The residues involved in the Mg²⁺ coordination (Glu⁹⁵, Gly¹²⁸, and Asp³¹⁸) are represented as sticks. C, molecular surface of ZgAgaC was calculated using PyMOL (version 1.8.2.2; Schrödinger, LLC, New York) and colored according to electrostatic local Coulomb potential ranging from deep blue (+ 66) to red (−66).

Figure 10.

Comparison of the active site of ZgAgaC and of the β-agarase ZgAgaB. A, superimposition of ZgAgaB_E189D (PDB code 4ATF, colored in cyan) in complex with an agarose octasaccharide (colored in gray; six moieties are shown) and ZgAgaC (colored in purple). B, schematic representation of the interaction of ZgAgaB_E189D with an agarose oligosaccharide. This figure was prepared with Ligplot version 4.5.3 (72). The labels of the ZgAgaC residues conserved with ZgAgaB are shown in blue. In both panels, the catalytic residues are underlined with a red line.

Figure 11.

Comparison of the active site of ZgAgaC and of the β-porphyranase ZgPorA. A, superimposition of ZgPorA_E139S (PDB code 3ILF, colored in yellow) in complex with a porphyran tetrasaccharide (colored in green) and ZgAgaC (colored in purple). B, schematic representation of the interaction of ZgPorA_E139S with the porphyran tetrasaccharide. This figure has been prepared with Ligplot version 4.5.3 (72). The labels of the ZgAgaC residues conserved with ZgPorA are shown in blue. In both panels, the catalytic residues are underlined with a red line.

Figure 12.

Details of the comparison of the active site of ZgAgaC, ZgAgaB (PDB code 4ATF) and ZgPorA (PDB code 3ILF). A, close-up view of the superposition of ZgAgaC, ZgAgaB, and ZgPorA, centered on the conserved tryptophans Trp¹¹⁰ (ZgAgaC, colored in purple), Zg109 (ZgAgaB, colored in cyan), and Trp⁵⁶ (ZgPorA, colored in yellow). Despite their conservation in sequence, these residues involved in substrate recognition are not spatially superimposed. B, close-up view of the superposition of ZgAgaC and ZgAgaB, centered on the −1 subsite. The glycerol molecule bound to ZgAgaC partially overlaps the d-galactose moiety bound to the −1 subsite in ZgAgaB.