Unraveling the molecular mechanism of polysaccharide lyases for efficient alginate degradation

Abstract

Alginate lyases (ALs) catalyze the depolymerization of brown macroalgae alginates, widely used naturally occurring polysaccharides. Their molecular reaction mechanism remains elusive due to the lack of catalytically competent Michaelis-Menten-like complex structures. Here, we provide structural snapshots and dissect the mechanism of mannuronan-specific ALs from family 7 polysaccharide lyases (PL7), employing time-resolved NMR, X-ray, neutron crystallography, and QM/MM simulations. We reveal the protonation state of critical active site residues, enabling atomic-level analysis of the reaction coordinate. Our approach reveals an endolytic and asynchronous syn β-elimination reaction, with Tyr serving as both Brønsted base and acid, involving a carbanion-type transition state. This study not only reconciles previous structural and kinetic discrepancies, but also establishes a comprehensive PL reaction mechanism which is most likely applicable across all enzymes of the PL7 family as well as other PL families.

Fig. 1. Alginate degradation.

a Stylized chemical structure of alginate. The unit blocks, α-l-guluronic acid and β-d-mannuronic acid, are colored orange and green, respectively. b Chemical reactions catalyzed by alginate lyases (ALs). The syn reaction is presented above, while anti reaction is shown below. Acid and base residues, which belong to the enzyme, are denoted as A and B, respectively, and colored in blue. c Structure representation of a PL7 (at the top, PDB 6YWF), a PL18 (in the middle, PDB 4Q8K), and a PL36 (at the bottom, PDB 6KCW) to show the shared jelly roll folding. A rotation of 90° over the Y axis highlights the characteristic solvent-exposed catalytic groove where they bind to the polymer.

Fig. 2. Time-resolved ¹³C-NMR spectra results of ¹³C1-enriched polyM treated with PsAlg7A or PsAlg7C.

Each reaction was conducted in 3 mm NMR tubes at 25 °C with 8.7 mg/mL enzyme in 170 μL of substrate buffer solution (10 mg/mL ¹³C1-polyM substrate in 5 mM Na-acetate, pD 5.5 with 100 mM NaCl and 1.5 mM ZnCl₂ in 99.9% D₂O) and 10 µL of 147.5 mg/mL PsAlg7A incubated at 25 °C. Pseudo 2D time-resolved spectra were recorded by acquiring 1D carbon spectra every 5 min for a total of 21 h 20 min. a shows the reaction for PsAlg7A, and b shows the reaction for PsAlg7C. MMM is the internal mannuronate in polyM substrate, and abbreviations of the products formed are: ΔM_n C/H-1 signal of 4-deoxy-l-erythro-4-hexenopyranouronate at the non-reducing end of a mannuronate oligomer/polymer, Mα C/H-1 signal of α reducing end of mannuronate oligomer/polymer, Mβ C/H-1 signal of the β reducing end of mannuronate oligomer/polymer, Δα signifies the C/H-1 signal of an α reducing end of mannuronate dimer with 4-deoxy-l-erythro-4-hexenopyranouronate at the non-reducing end.

Fig. 3. PsAlg7A and C active site architecture.

a PsAlg7A (light purple) soaked with PentaM (light green), b PsAlg7A-Y223F (light purple) soaked with HexaM (green), c PsAlg7C (cyan) soaked with HexaM (M moieties: dark green; G moieties: orange), and d PsAlg7C-Y220F (cyan) soaked with HexaM (M moieties: dark green; G moieties: orange) (NR and R indicate the non-reducing end and reducing end, respectively). The observed AOSs are longer than the AOSs used for soaking likely because they are accommodated in two or more different ways in the active sites. e Electrostatic plot of PsAlg7A at pH 5 and f electrostatic plot of PsAlg7C at pH 8 with electron density maps, 2Fo-Fc (blue mesh) contoured to 1.0σ with a cutoff at 1.6 Å for C and fc-fo (green and red) contoured to +3.0 and −3.0σ, respectively, with a cutoff at 1.6 Å for C.

Fig. 4. PsAlg7C-H124N (cyan) soaked with PentaM (dark green).

a Active site groove and b the surface binding site (SBS). Amino acid residues are shown as sticks, highlighting N124 conformations in (a) and residues within 4 Å of the tetrasaccharide in (b). The G moiety in (a) is colored in orange.

Fig. 5. Organization of binding sites in the presence and absence of the ligand.

a The apo structures of PsAlg7A and b PsAlg7C exhibit conserved water molecules and residue orientations, c which are compared with the PsAlg7A-Y223F and d PsAlg7C-Y220F mutants soaked with HexaM. For simplicity, only sugars at subsites −1 and +1 are shown. Conserved water molecules, residues, and putative hydrogen bonds formed with the M moieties at subsites −1 and +1 are highlighted in the superimposed apo and complex structures for e PsAlg7A and f PsAlg7C. Yellow dotted lines indicate putative hydrogen bonds.

Fig. 6. Protonation states of putative catalytic residues.

Structure of a PsAlg7C, b PsAlg7C soaked with PentaM and c superimposition of both. Nuclear density map 2Fo-Fc (blue mesh and magenta mesh for apo and soaked, respectively) contoured at 0.5σ with a cutoff at 1.6 Å. The disordered deuterium was tentatively placed in a riding position on the hydroxyl group of Y220.

Fig. 7. Computational modeling of the syn β-elimination reaction catalyzed by PsAlg7A.

a Collective variables used in the QM/MM OPES simulations of the catalytic mechanism of PsAlg7A. b Free energy landscape (FEL) obtained from the simulations and minimum free energy pathway defining the reaction coordinate. c Potential Mean Force (PMF) along the minimum energy pathway. d Evolution of the most relevant distances along the reaction coordinate. e Evolution of the Mulliken charges of the main atoms involved in the catalysis of sugar at subsite +1 during the reaction. Data are presented as mean values ± SD, obtained from unbiased QM/MM MD simulations, as described in the methods section (nR = 2000, nTS = 50 and nP = 2000).

Fig. 8. Molecular changes along the reaction coordinate in PsAlg7A.

a Representative structures along the reaction coordinate, obtained from QM/MM OPES simulations. Hydrogen atoms that are attached to C atoms have been omitted for clarity, except the H5 atom. b Proposed reaction mechanism for PsAlg7A according to all results of this work.