Structural insights and rational design of Pseudomonas putida KT2440 Omega transaminases for enhanced biotransformation of (R)-Phenylacetylcarbinol to (1R, 2S)-Norephedrine
Omega transaminases ({omega}-TAs) can mediate the chiral amination of several unnatural substrates without the requirement of an -COOH group, and are highly relevant in the production of several pharmaceutical intermediates of commercial interest. Development of better variants of {omega}-TAs are hence essential for their industrial uses. We have studied the active site architecture of the wild-type {omega}-TAs, to develop engineered enzymes for enhancing the biotransformation of (R)-Phenylacetylcarbinol to (1R, 2S)-Norephedrine. Two such {omega}-TAs (TA_5182 and TA_2799) from P. putida KT2440 strain were overexpressed and purified as recombinant proteins. Crystal structures of TA_5182 were solved in two conformations, and significant movements of two highly flexible loops were observed in these different states. The TA_2799 structure was determined in the co-factor bound state with a PLP molecule covalently bonded to the catalytic K286 as an internal aldimine. Enzyme assays indicated that TA_2799 required significantly higher concentrations of co-factor than TA_5182 to achieve satisfactory biotransformation of (R)-PAC. A key mutation of L322F in TA_2799 drastically reduced the co-factor dependency of the TA_2799_L322F mutant enzyme, and the mutant remained active for 96h at 30{degrees}C. The crystal structure of the mutant enzyme revealed an asparagine residue that mediates a hydrogen bonding network at the dimeric interface of the enzyme and is absent in TA_5182. The TA_5182_G119N mutant also showed enhanced co-factor affinity. The results of our studies will help generate Pseudomonad {omega}-TAs and {omega}-TAs from other organisms with high efficiency for asymmetric synthesis, to be used in host systems for optimal large-scale industrial biotransformation.