6S54

Transaminase from Pseudomonas fluorescens

Summary for 6S54

• Entry DOI: 10.2210/pdb6s54/pdb
• Descriptor: Aspartate aminotransferase family protein, PYRIDOXAL-5'-PHOSPHATE, GLYCEROL, ... (5 entities in total)
• Functional Keywords: class-i omega-transaminase, plp, transaminase, pyridoxal phosphate, transferase
• Biological source: Pseudomonas fluorescens
• Total number of polymer chains: 4
• Total formula weight: 211748.91

Authors

Smith, P.,Roura Padrosa, D.,Lopez-Gallego, F.,Paradisi, F.,Dreveny, I. (deposition date: 2019-06-30, release date: 2019-11-13, Last modification date: 2024-01-24)

Primary citation

Roura Padrosa, D.,Alaux, R.,Smith, P.,Dreveny, I.,Lopez-Gallego, F.,Paradisi, F.
Enhancing PLP-Binding Capacity of Class-III omega-Transaminase by Single Residue Substitution.
Front Bioeng Biotechnol, 7:282-282, 2019

PubMed Abstract: Transaminases are pyridoxal-5'-phosphate (PLP) binding enzymes, broadly studied for their potential industrial application. Their affinity for PLP has been related to their performance and operational stability and while significant differences in PLP requirements have been reported, the environment of the PLP-binding pocket is highly conserved. In this study, thorough analysis of the residue interaction network of three homologous transaminases (HeTA), (CvTA), and (PfTA) revealed a single residue difference in their PLP binding pocket: an asparagine at position 120 in HeTA. N120 is suitably positioned to interact with an aspartic acid known to protonate the PLP pyridinium nitrogen, while the equivalent position is occupied by a valine in the other two enzymes. Three different mutants were constructed (HeTA-N120V, CvTA-V124N, and PfTA-V129N) and functionally analyzed. Notably, in HeTA and CvTA, the asparagine variants, consistently exhibited a higher thermal stability and a significant decrease in the dissociation constant ( for PLP, confirming the important role of N120 in PLP binding. Moreover, the reaction intermediate pyridoxamine-5'-phosphate (PMP) was released more slowly into the bulk, indicating that the mutation also enhances their PMP binding capacity. The crystal structure of PfTA, elucidated in this work, revealed a tetrameric arrangement with the PLP binding sites near the subunit interface. In this case, the V129N mutation had a negligible effect on PLP-binding, but it reduced its temperature stability possibly destabilizing the quaternary structure.

PubMed: 31681755
DOI: 10.3389/fbioe.2019.00282

Experimental method

X-RAY DIFFRACTION (2.21 Å)