The bacterial ω‐transaminase from Chromobacterium violaceum (Cv‐ωTA, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/6/1/18.html) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5′‐phosphate (PLP). Here, we present four crystal structures of Cv‐ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo‐Cv‐ωTA structure reveals unique ‘relaxed’ conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered ‘roof ’ over the PLP‐binding site, whereas in the other apo form and the holo form the ‘roof’ is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the ‘roof’ structure may be associated with substrate binding in Cv‐ωTA and some other transaminases. Database The atomic coordinates and structure factors for the Chromobacterium violaceumω‐transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4A6U for the holoenzyme, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4A6R for the apo1 form, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4A6T for the apo2 form and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4A72 for the mixed form Structured digital abstract  http://www.uniprot.org/uniprot/Q7NWG4 and http://www.uniprot.org/uniprot/Q7NWG4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0038 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8300874)  http://www.uniprot.org/uniprot/Q7NWG4 and http://www.uniprot.org/uniprot/Q7NWG4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8300763)  http://www.uniprot.org/uniprot/Q7NWG4 and http://www.uniprot.org/uniprot/Q7NWG4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8300950)

Enantiopure chiral amines synthesis using omega-transaminases is hindered by an unfavourable equilibrium, but when using isopropylamine as the amine donor the equilibrium can be completely displaced by using a specific dehydrogenase in situ for removal of formed acetone.

Transaminases inherently possess high enantiospecificity and are valuable tools for stereoselective synthesis of chiral amines in high yield from a ketone and a simple amino donor such as 2‐propylamine. Most known ω‐transaminases are (S)‐selective and there is, therefore, a need of (R)‐selective enzymes. We report the successful rational design of an (S)‐selective ω‐transaminase for reversed and improved enantioselectivity. Previously, engineering performed on this enzyme group was mainly based on directed evolution, with few exceptions. One reason for this is the current lack of 3D structures. We have explored the ω‐transaminase from Chromobacterium violaceum and have used a homology modeling/rational design approach to create enzyme variants for which the activity was increased and the enantioselectivity reversed. This work led to the identification of key amino acid residues that control the activity and enantiomeric preference. To increase the enantiospecificity of the C. violaceum ω‐transaminase, a possible single point mutation (W60C) in the active site was identified by homology modeling. By site‐directed mutagenesis this enzyme variant was created and it displayed an E value improved up to 15‐fold. In addition, to reverse the enantiomeric preference of the enzyme, two other point mutations (F88A/A231F) were identified. This double mutation created an enzyme variant, which displayed substrate dependent reversed enantiomeric preference with an E value shifted from 3.9 (S) to 63 (R) for 2‐aminotetralin.

For biocatalytic production of pharmaceutically important chiral amines the ω-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum ω-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (∼5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.

A pilot-plant-process is described where frakefamide × HCl (L-tyrosyl-D-alanyl-p-fluoro-L-phenylalanyl-L-phenylalaninamide hydrochloride) was synthesised from its amino acid monomers in seven steps. The synthesis was performed in 70-L equipment, and the final product was obtained in 70% overall yield and in 99.5% purity. Only two intermediates were isolated, and the process required no chromatography. Peptide bond formation was promoted by isobutyl chloroformate-mediated mixed anhydride coupling reactions. The formed mixed anhydrides proved to be surprisingly stable, in most cases for several hours at -10 °C, and therefore suitable for large-scale peptide synthesis. Only traces, if any, of racemised coupling products were obtained. Benzyloxycarbonyl was used as amino protecting group throughout the synthesis, and its removal by hydrogenolysis proved to be fast and convenient on a large scale.