| Tyrosine decarboxylase(TDC) is a pyridoxal 5-phosphate(PLP) dependent enzyme and mainly responsible for the synthesis of tyramine from the decarboxylation of tyrosine. Tyramine and its derivatives are important and key intermediates of many high value-added products, such as pharmaceuticals, health products, and cosmetics etc. With the deep understanding of the novel activities of tyramine, the green and efficient synthesis is of special interest. However the detailed catalytic mechnism and key residues of TDC were unclear. As a result, recombinant expression of TDC from Lactobacillus brevis, purification, crystallization, X-ray diffraction, structural analysis and site directed mutagenesis on active sites were carried out in this study. The main results are listed as follows:Firstly, the structure of TDC from Lactobacillus brevis was resolved. This recombinant TDC was expressed with Se-Methionine to form Se-TDC and purified to homogeneity. Purified Se-TDC was used for crystallization by sitting-drop-vapor-diffusion method. Diffraction data of Se-TDC was collected at High Energy Accelerator Research Organization(KEK, Japan). The structure of apo-TDC was solved by multiwavelength anomalous diffraction at 1.73 ?(PDB: 5HSI). The structure of TDC in complex with coenzyme PLP(holo-TDC) was also obtained by molecular replacement at 1.90 ?(PDB: 5HSJ). Comparison of apo- and holo-TDC structures revealed that there are two opposite conformations of K240 and H241. In the holo-TDC, the imidazole ring of H241 swings to the pyridine ring of PLP while it is distant from the PLP binding pocket in the apo-TDC. K240 A and semi-saturation mutagenesis on H241 were performed. The kcat/Km of K240 A towards tyrosine and DOPA decreased and the Km of H241 mutants increased significantly, as compared to wild-type TDC. Consequently H241 plays an important role in the binding of coenzyme and substrate, whereas K240 A might affect the affinity of enzyme and substrates by alteration the conformation of adjacent amino acids, especially H241.Secondly, the role of residues in the active site was investigated by site-directed mutagenesis. Substrate tyrosine was docked into the holo-TDC,and a number of residues around the putative substrate binding pocket were selected for site-directed mutagenesis study. Alanine scanning showed several residues affecting the activity significantly, including E102, Y395, Y398 and S586 etc. Semi-saturation mutagenesis on E102 and Y395 showed minimal change in activity when mutated into Asp and Phe with similar structures and properties, and they have 67.9% and 60.2% activity compare with WT, while other mutants showed less than 10%. They may play important role in the stabilization of spatial conformation at active site of TDC by salt bridge and aromatic stacking. The Km of variants Y398 A and Y398 F towards tyrosine is 5.14 and 4.07 folds higher than that of WT., and the kcat of Y398 F and Y398 A towards tyrosine is significantly decreased. These demonstrate that Y398 is important for the catalysis and binding of amino acid substrate. S586 A displays over 2.23 folds improvement in activity compared with that of WT(96.0 U·mg–1 versus 43.1 U·mg–1), representing the highest activity among all tested variants. The kcat and Km of S586 A towards tyrosine are 251 s–1 and 0.42 mmol·L–1, while the kcat/Km of S586 A towards tyrosine is 600 L·s–1·mmol–1, which is 2.78 folds of WT. Saturation mutagenesis on S586 was performed to elucidate its detailed mechanisms. Besides S586 A, only S586 G and S586 T retained over 10% activity toward tyrosine, while other mutants showed less than 5% or undetectable activity on tyrosine. It is speculated that there are specific requirement on the steric hindrance and hydrophobicity of residue 586. Our results provide structural basis and interesting residues for the protein engineering on catalytic efficiency of TDC for green and efficient preparation of tyramine. |
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