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 Halomonas elongata (HeTA), Chromobacterium violaceum (CvTA), and Pseudomonas fluorescens (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 (Kd) 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.
Highlights
Pyridoxal 5′-phosphate (PLP) is the biologically active form of vitamin B6 and plays a key role in the enzymatic mechanism of a variety of biochemical reactions (Percudani and Peracchi, 2003; Eliot and Kirsch, 2004)
Crystals were flash cooled after soaking in cryoprotectant solution containing six parts precipitant mixture with four parts glycerol. Diffraction data for these crystals were collected on the I24 beamline at the Diamond Light Source and the structure was solved by molecular replacement using coordinates from the uncultivated Pseudomonas sp. transaminase (PDB: 5LHA) as a search model with PHASER
The CvTAV124N new variant showed a dissociation constant (Kd) comparable to that of WTHeTA and WT-Pseudomonas fluorescens ω-transaminase (PfTA). These results suggest that N120 of Halomonas elongata (HeTA) reinforces the interaction network of the acidic residues that anchors the pyridinium ion of PLP (Figure 4A)
Summary
Pyridoxal 5′-phosphate (PLP) is the biologically active form of vitamin B6 and plays a key role in the enzymatic mechanism of a variety of biochemical reactions (Percudani and Peracchi, 2003; Eliot and Kirsch, 2004). One of the most important groups among PLP-dependent enzymes are transaminases (TAs). The reaction starts with a holo-enzyme complex where PLP is initially bound to the enzyme through an imine bond with the ε-NH2 of the catalytic lysine (internal aldimine). This complex reacts with the amine donor forming an external aldimine that after hydrolysis gives rise to pyridoxamine 5′-phosphate (PMP) and the deaminated product. If PMP remains at the active site, it readily interacts with the amine acceptor to form the aminated product and returns to the holo-form of the enzyme (PLP-bound) to start a new catalytic cycle (Figure 1)
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