Fig 1.
Three-dimensional structure of vanillyl alcohol oxidase (PDB ID: 1VAO).
The dimer is shown as cartoon with the cap domain coloured in red and FAD-binding domain in green. A zoom into the active site at the interface of the two domains is shown with selected residues shown as blue sticks and the isoalloxazine ring of the covalently bound flavin cofactor in yellow.
Fig 2.
Overview of the reaction cycle from p-creosol to vanillyl alcohol and vanillin catalysed by VAO.
Reaction rates at pH 7.5, 25°C are shown in the centre of the reaction cycle (data obtained from [22]). The rate-limiting step for the conversion of p-creosol to vanillyl alcohol is the decomposition of the air-stable flavin N5 substrate adduct (panel 4, marked in red). For the conversion of vanillyl alcohol to vanillin the rate-limiting step is the reduction of the flavin by the substrate (panel 7). In both reactions, the yellow flavin cofactor is rendered colorless through reduction by the substrate (occurring between panels 2 and 3 as well as 6 and 7). Molecular oxygen driven oxidation of reduced flavin (colourless) to oxidized flavin (yellow) occurs between panels 5 and 6 as well as panels 8 and 1, in the presence or absence of either vanillyl alcohol or vanillin.
Fig 3.
Three-dimensional structure of vanillyl alcohol oxidase and the four paths identified in this study.
Vanillyl alcohol oxidase is shown as cartoon and coloured in grey, with FAD shown as sticks and coloured in yellow. (PDB ID: 1VAO). The paths identified in this work are shown through sphere representation of ligand positions in the trajectories. The cap path is coloured in orange, the FAD path in blue, the subunit interface path in magenta and the re path in cyan. A: Octameric VAO, seen from the front. B: Octameric VAO seen from the side. C: Dimeric VAO as seen in Fig 1.
Fig 4.
Overview of the entrance path identified with COP (A) as well as RMSF and atom contacts (B, for COP) and binding energy data (C, for COP and COD). A: The VAO dimer is shown as cartoon in grey and its FAD cofactor is shown as sticks in yellow. The entrance path is shown through sphere representation of ligand positions in the trajectories and coloured in magenta. For clarity, we only show data for an entrance simulation where COP migrates to the left-hand subunit. B: Highest RMSF values (black boxplot boxes) and atom contact counts with the ligand (grey boxplot boxes) of residues in VAO. Magenta filling of the boxplots indicates that the residue belongs to the entrance path. Residues that do not have atom contacts with the ligand but are highly flexible only have a black boxplot (for RMSF). Because data was obtained from only one simulation, the atom contacts do not have any variation. C: Scatterplot of binding energies as a function of the distance between the ligand and FAD. Negative x-values indicate the distance to the FAD in the other subunit. This graph shows that the path is symmetrical for COP (in magenta).
Fig 5.
Overview of the exit paths identified with COP and COD (A), RMSF and atom contacts (B for COP and C for COD) and binding energies (D). A: The VAO dimer is shown as cartoon in grey and its FAD cofactor is shown as sticks in yellow. The paths identified in this work are shown through sphere representation of ligand positions in the trajectories. The cap path is coloured in orange, the FAD path in blue and the subunit interface path in magenta. B: Plot of simulations with COP, highest RMSF values (black boxplot boxes) and atom contact counts with the ligand (grey boxplot boxes) of residues in VAO. Coloured filling of the boxplots indicates that the residue belongs to the path of that colour. C: Plot of simulations with COD, highest RMSF values (black boxplot boxes) and atom contact counts with the ligand (grey boxplot boxes) of residues in VAO. Coloured filling of the boxplots indicates that the residue belongs to the path of that colour. D: Scatterplot of binding energies as a function of the distance between the COP and COD (in green and black) to FAD.
Fig 6.
Details of the ligand migration paths identified in this study (A) and illustration of the surface at the re path in VAO, EUGO and PCMH (B). A: Crystal structures of VAO, EUGO and PCMH as well as selected frames from our simulations were aligned using PyMOL. Selected crystal structure residues (labelled with VAO numbering) are shown as sticks. Residues involved in the subunit interface path and their conformations in VAO are coloured in magenta. The conformations of the corresponding residues in EUGO and PCMH, are coloured in lighter or darker shades of magenta, respectively. Residues involved in the re path and their conformations in VAO are coloured in cyan. The conformations of the corresponding residues in EUGO and PCMH, are coloured in lighter or darker shade of cyan, respectively. Note that the loop carrying Tyr51 is completely absent in EUGO and PCMH and that Tyr408 present in VAO is a leucine in EUGO and PCMH. Selected residues from selected frames of our simulations are shown as lines, in grey. Frames showing the movement of Tyr51 and Tyr408 were taken from simulations with dioxygen, those showing movement of His466, Tyr503 and Met195 from simulations with phenolic ligands that were migrating through the subunit interface path. The respective other monomer of the three crystal structures is visible on the right of the figure as cartoon in red (cap domain) and green (FAD-binding domain). On the left, the cytochrome c subunit from PCMH is visible as cartoon, coloured in blue. B: Surface representation of the surface of the re path to illustrate the size of the channel leading to the re side of FAD in VAO, EUGO and PCMH. The proteins are shown as surfaces and the FAD cofactors as sticks, coloured in yellow. Note the different size of the channel in VAO and EUGO, as well as the blockage by the cytochrome c subunit in PCMH (blue mesh).
Fig 7.
Overview of the re path identified with dioxygen (OXO) and hydrogen peroxide (PER) (A), RMSF and atom contacts (B for dioxygen and C for hydrogen peroxide) and binding energies (D). A: The VAO dimer is shown as cartoon in grey and its FAD cofactor is shown as sticks in yellow. The paths identified in this work are shown through sphere representation of ligand positions in the trajectories. The re path is coloured in cyan. B: Plot of simulations with dioxygen, highest RMSF values (black boxplot boxes) and atom contact counts with the ligand (grey boxplot boxes) of residues in VAO. Cyan filling of the boxplots indicates that the residue belongs to the re path. C: Plot of simulations with hydrogen peroxide, highest RMSF values (black boxplot boxes) and atom contact counts with the ligand (grey boxplot boxes) of residues in VAO. Coloured filling of the boxplots indicates that the residue belongs to the path of that colour. D: Scatterplot of binding energies as a function of the distance between dioxygen (cyan) or hydrogen peroxide (black) to FAD.
Fig 8.
Structure-based sequence alignment of VAO, EUGO and PCMH.
Numbering of residues at the beginning and end of each line correspond to the numbering used in the crystal structures. Numbers on top of the alignment correspond to the numbering of VAO sequence P56216.1 and is the same as used throughout this study. Coloured symbols indicate the path the residue underneath it is involved in. Orange, blue, magenta and cyan was used for the cap, the FAD, the subunit interface and the re path respectively. Red indicates functionally important residues. Bi-coloured symbols indicate the residue is involved in two paths. The shape of the symbol indicates preservation of the residue, stars (present in all three structures), circles (present in two of the three structures) and squares (present in only one of the three structures).
Fig 9.
Sequence preservation of ligand migration paths.
Preservation of residues in the entire protein, a random selection (random), the cap domain, the subunit interface, on the surface of the protein (surface), in the cap path, the FAD path, the subunit interface path and the re path. A: Sequence preservation of ligand migration paths within MSAVAO, MSAEUGO and MSAPCMH. B: Sequence preservation of ligand migration paths within MSAall.
Fig 10.
Overview of the identified paths and the ligands proposed to migrate through them (uni-or bi-directional).
The VAO dimer is shown in surface representation, with the cap domain coloured in red and the FAD-binding domain coloured in green and the FAD cofactor shown as sticks in yellow. Coloured arrows indicate the paths for phenolic ligands (cap path in orange, FAD path in blue and subunit interface path in magenta). Filled in arrowheads indicate paths to the si side of FAD and empty arrowheads paths to the re side of FAD. Drawn out lines indicate the path is on the side of VAO facing the viewer, and dotted lines indicate the path is on the back side of VAO. Note that the two subunits are rotated 180 degrees relative to each other in the VAO dimer and that a path pointing towards the viewer in the right monomer is pointing away from the viewer in the left monomer and vice versa. The location of the concierge residues, His466 and Tyr503, is represented by a black line. The portal formed by Met192, Met195 and Glu464 is indicated by an ellipse. The bottlenecks formed by Arg312 and Arg398 in the cap path and Trp89 and Lys5454 in the FAD path are represented by pentagrams and heptagrams, respectively.