9J4Z
Crystal structure of the open state of omega transaminase TA_5182 from Pseudomonas putida KT2440
Summary for 9J4Z
| Entry DOI | 10.2210/pdb9j4z/pdb |
| Descriptor | Polyamine:pyruvate transaminase, SULFATE ION (2 entities in total) |
| Functional Keywords | omega transaminase, aminotransferase, thermal stability, pseudomonas putida, cofactor affinity, transferase |
| Biological source | Pseudomonas putida KT2440 |
| Total number of polymer chains | 4 |
| Total formula weight | 200981.63 |
| Authors | Das, P.,Bhaumik, P. (deposition date: 2024-08-10, release date: 2025-06-04, Last modification date: 2025-06-11) |
| Primary citation | Das, P.,Noronha, S.,Bhaumik, P. Structural insights and rational design of Pseudomonas putida KT2440 Omega transaminases for enhanced biotransformation of (R)-PAC to (1R, 2S)-Norephedrine. J.Biol.Chem., :110289-110289, 2025 Cited by PubMed Abstract: Omega transaminases (ω-TAs) can mediate the chiral amination of several unnatural substrates without the requirement of an α-COOH group and are highly relevant in the production of several pharmaceutical intermediates of commercial interest. Development of better variants of ω-TAs is hence essential for biotransformation of unnatural substrates. We studied the active site architecture of the wild-type ω-TAs, to engineer enzymes that enhance the biotransformation of (R)-phenylacetylcarbinol to (1R, 2S)-norephedrine. Two such ω-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 as a complex with the cofactor pyridoxal 5'-phosphate (PLP) covalently bound to the catalytic K286 as an internal aldimine. Enzyme assays indicated that TA_2799 required a four-fold higher cofactor concentration than TA_5182 to achieve satisfactory biotransformation of (R)-PAC. A key mutation of L322F in TA_2799 drastically reduced (∼8-fold) the cofactor dependency of the TA_2799_L322F mutant enzyme, and the mutant remained active for 96 h at 30°C. The crystal structure of the mutant enzyme revealed a key 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 cofactor affinity. The results of our studies will help generate Pseudomonad ω-TAs and ω-TAs from other organisms with high efficiency for asymmetric synthesis, for further applications in large-scale biotransformation processes. PubMed: 40436318DOI: 10.1016/j.jbc.2025.110289 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (3.4 Å) |
Structure validation
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