Heterologous Expression And Characterization Of Homoserine Dehydrogenase Mutants Incorynebacterium Pekinense

Aspartic acid family amino acids (lysine, threonine, methionine)are essential for animalsand humans, and are synthesized with aspartic acid as precursor in a series of complexmetabolic pathways in microorganisms system but can not in human body. Homoserinedehydrogenase (HSD), which is one key enzyme in the biosynthesis pathway of aspartate aminoacid, plays an important role in the biosynthesis of methionine, threonine and isoleucine. Inorder to improve the yield of methionine and threonine, it is important to study the catalyticmechanism of key enzymes in this pathway and the modification of the protein structurerationality, for feedback inhibition or feedback repression by the end product. In this study, theeffect of specific amino acid sites of HSD from Corynebacterium pekinense on enzyme activityhas been analysed through bioinformatics and modern biotechnology.1. Through BLAST sequence alignment,3MTJ, which was from Thiobacillus Denitrificans,was chosen as template for modeling for40%homology withHSD from C. pekinense bymodelling software Insight II97. The active site and the key associated amino acid residues incrystal structure were found by PyMOL, advanced molecular docking program Affinity wasused to simulate molecular docking with substrate; and conservatism of the key residues wereanalysed by Clustalw sequence alignment.D215was chosen to be mutanted for its polarity effectwith the active site.2. Four HSD single mutants D215A, D215E, D215G and D215K were constructed andefficiently expressed in Escherichia coli BL21, enzyme kinetics and the enzymatic properties ofthe purified mutant proteins were determined. The results showed that: the fitting of kineticcurve revealed that Vmax of WT, D215A, D215E, D215G and D215K were18.46U/mg,13.09U/mg,23.57U/mg,24.29U/mg,16.19U/mg, respectively;as shown on the kinetic parameters,the mutant D215E and D215K showed unique dynamic characteristics, both had higher catalyticefficiency with larger Vmax and smaller Km compared with wild-type; mutant D215A had abetter substrate affinity compared with the wild-type; n value of mutant D215G was close to1tending to Michaelis enzyme. The optimum temperature of WT, D215A, D215E, D215G andD215K were37℃,37℃,40℃,35℃,37℃, respectively. The optimum pH were7.5,7.5,8.0,8.0,8.0, respectively. Half-life of HSD were3.8h,5.38h,7.62h,2.58h and4.76h,respectively.The ethanol had an effect of activation on mutants at the concentration of5%. 3.To further improve the enzyme activity, the double mutants L200F-D215A,L200F-D215E,L200F-D215G, L200F-D215K were successfully constructed by the mutation ofD215on the basis of pET-L200F. The enzyme kinetics results showed that: the Vmax ofL200F-D215A, L200F-D215E, L200F-D215G, L200F-D215K were78.13%，97.88%，88.24%and124.39%of that of WT, respectively. The Km of L200F-D215A, L200F-D215E,L200F-D215G, L200F-D215K were80.58%，88.28%，81.50%and91.26%of that of WT,respectively. L200F/D215K was chosen to characterize for its highest catalytic efficiency, theresults showed that: The optimum pH was7.5; the optimum temperature was37℃; half-lifewas4.16h;Mg2+promoted the enzymetic activity at the concentration of1mmol/L, and also ithad good resistance to organic solvents. This study provides the experimental evidence for themolecular mechanism of HSD and the half-rational reconstruction of protein in the future.