Enzymatic synthesis of benzylisoquinoline alkaloids using a parallel cascade strategy and tyrosinase variants

Benzylisoquinoline alkaloid derived pharmaceuticals are widely applied in modern medicines. Recent studies on the microbial production of benzylisoquinolines have highlighted key biological syntheses towards these natural products. Routes to non-natural benzylisoquinolines have been less explored, particularly halogenated compounds which are more challenging. Here, we show the use of a tyrosinase, tyrosine decarboxylase, transaminase, and norcoclaurine synthase which are combined in a parallel cascade design, in order to generate halogenated benzylisoquinoline alkaloids in high enantiomeric excess. Notably, mutagenesis studies are applied to generate tyrosinase mutants, which enhance the acceptance of halogenated tyrosines for use in the biocatalytic cascades developed.


diol (S)-57
To synthesize the amine moiety, reaction mixture A (RMA, 20 mL, pH 5) consisted of 50   Supplementary Fig. 2 Images of SDS-PAGE electrophoresis for the protein expression of proteins used in the study. For enzyme purification: Lane 1: cell lysates; Lane 2: flow through with cell lysates; Lane 3: Flow through with wash buffer (containing 20 mM imidazole); Lane 4: Purified protein. Protein markers for a, b,

Achiral analytical HPLC results for CnTYR and EfTyrDC reaction products
Achiral separation was achieved using Analytical HPLC method 1. Pink represents HPLC trace of 3-Cl-L-DOPA 44 (the first peak) and 12 (the second peak). Blue represents HPLC trace of 3-Cl-dopamine (the first peak) and 3-Cl-tyramine (the second peak).

Preparing your protein
Using Chimera (UCFS Chimera version 1.13.1), the protein PDB was loaded and any water molecules and other subunits deleted. Selenomethionine residues were mutated to methionine and selenocysteine to cysteine. The protein was then saved as a pdb file. The protein pdb file was opened using AutoDock Tools (version 1.5.6) and hydrogens added to the protein. The box size for ligand docking was set to cover the whole protein (to encompass the active site, but not bias the results) and saved as a pdbqt file.

Preparing the ligand
ChemDraw smiles were generated and the energy of the ligand minimised using Avogadro (version 1.2.0). Again, the ligand pdb file was converted into a pdbqt file via AutoDock Tools.

Using Autodock Vina (v.1.2.0)
A text file was created input all the parameters for docking: receptor = protein file name.pdbqt ligand = ligand file name.pdbqt out = out.pdbqt center_x = 21.023 center_y = 11.027 center_z = 11.504 size_x = 40 size_y = 40 size_z = 40 Center_x,y and z and the size parameters were for the docking box -the values that were recorded from autodock tools. 'out = out.pdbqt' stored output files (different conformations and positions of the ligand). This was saved as the receptor and ligand pdbqt files. Then docking was performed via Vina through a terminal window. Vina was run and the binding modes results and energies were saved in a log file (log.txt).

Viewing the results
Docking results (binding modes) were viewed using Chimera by loading in the receptor pdb file and then out.pdb (all the output files). Nine possible binding modes were generated and ranked according to the affinity free energy. The scoring function used in Vina was derived using the PDBbind data set, in which the receptors were treated as rigid components, and the ligands as flexible molecules with the number of active rotatable bonds ranging from 0 to 32. Vina uses a gradient optimization method in its local optimization procedure. The calculation of the gradient gives the optimization algorithm a "sense of direction" from a single evaluation. By using multithreading, Vina can further speed up the execution by taking advantage of multiple CPUs or CPU cores. 7  12 using the crystal structure of BmTYR (PDB code: 3NPY) 11 as a template. b. The ranking order followed the affinity energy. c. Orientation state (see below): If Yes, the para/meta-OH on the ligand aromatic ring binds to CuA, meanwhile the near carbon of para/meta-OH is orientated towards CuB. Otherwise, the ligand is not in a suitable orientation state. Docking was performed by Autodoc vina. 7 Images were generated using Chimera. [8][9][10] d. Distance 1: the para/meta-OH on the ligand aromatic ring to CuA of CnTYRs. e. Distance 2: the near carbon of para/meta-OH on the ligand aromatic ring to CuB of CnTYRs. Out ---

Protein sequence alignment for CnTYR, RsTYR and BmTYR
a CnTYR and BmTYR compound 12 to adopt a productive orientation; The blue balls represent the di-copper centre of CnTYR. The functional histamine residues in the tyrosinase active sites and substrates are shown in stick and ribbon forms. meta-L-tyrosine 9 is shown in rose and Cl-L-tyrosine 12 is shown in light-blue. Enzymes are shown in tan. Using the crystal structure of BmTYR (PDB code: 3NPY) 11 as a template, homology modelling (SWISS-MODEL) 12 was used to develop a model of WT-CnTYR. CnTYR variants were homology modelled by Chimera. Asp201 is shown in dark-grey and Ser201 is shown in orange. CnTYR variants were homology modelled by Chimera 9 . Figures were generated by Chimera. 8,10 Supplementary Fig. 91 Yields of the halogenated alkaloids (S)-45 and (S)-48 using TfNCS variants. Orange bars represent the yields of the fluorinated alkaloid (S)-45. Grey bars represent the yields of the chlorinated alkaloid (S)-48 using the wildtype TfNCS and variants L76V, A79I, A79F, F80L, M97F and A182I. The 100% product yield refers to the theoretical yield. Experiments were performed in triplicates, and measurements were taken from distinct samples. Error bars indicate the standard error of the triplicates.
Global docking protocol for TfNCS variants and intermediates 58/59 was the same as described in SI11. The grid box size for proteins was as follows:   a The ranking order followed the affinity energy. b Conformation state of the heterocyclic ring. c Distance: the distance between the iminium carbon of both 58 and 59 and the carbon ortho to the F/Cl on the aromatic ring.