Site-directed Mutagenesis And Functional Analysis From Corynebacterium Pekinense Aspartate Kinase

Aspartate kinase(AK) is a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, and its activity is strictly regulated by metabolites which can effect the yield of aspartate-derived amino acids. This study aimed to improve the activity of AK and relieve feedback inhibition for some metabolites of AK so that obtaining a high yield of aspartate-derived amino acids. Specific methods: Based on the template of pET-28a-AK, mutations with high activity were constructed successfully by site-directed mutation, as shown by enzymetic characterization. Researches and the results are as follows:1.By blast sequence alignment, the sequence homology of Corynebacterium pekinense AK(Cp-AK) and Corynebacterium glutamicum AK(Cg-AK) is 99%, sharing a similar α2β2-type structure. In this study, we used the crystal structure of Cg-AK as the template and sequenced alignment to identify part of conserved residues in regulatory domain of α subunit in Cp-AK, which formed an interconnected network mediated mainly by Van der Waals interactions or hydrogen bonds that can steady the lysine-binding site. We speculated that destroying the interaction between these residues and the lysine-binding site may partly deregulate Cp-AK from allosteric inhibition by lysine and threonine. To test these hypotheses, we selected Cp-AK M372, G377 and V378 for mutagenesis studies which was conserved in α subunit of the regulatory domain of AK. And then selecting mutation strains with high activity by HTS. M372 R, M372 G, M372 D, M372 I, G377 D, G377 K, G377 F, G377 Y, V378 F, V378 D and V378 H were obtained. And then those strains were expressed in E.coli BL21. AKs’ molecular weight was 48 kDa, as shown by the result of SDS-PAGE and Western blotting.2. The kinetic parameters Vmax of mutant M372 D, M372 G, M372 I and M372 R AK were 7.74, 6.54, 33.59 and 3.86 times higher than that of WT AK, respectively. The n value of the mutation AK was 1.1, 1.5, 1.9 and 1.6, respectively. Indicating the positive cooperativity of AK decreased. Thus, chosing M372 I AK to do the further study. As shown by enzymetic characterization, the optimum reaction pH of M372 I was 8.0, which was slightly lower than that of WT(pH 8.5). The optimum reaction temperature of M372 I was 30?C, which was higher than that of WT(25?C). Under the optimum reaction temperature and pH, the half-life period of M372 I increased from 2.7h(WT) to 4.7h. K+ in high concentration(10mM), the relative enzymatic activity was 176.8±4.1%. However, Mg2+, Mn2+ and Fe3+ in low concentration(0.2mM) the relative enzymatic activity was 154.9±4.3%, 132.9±2.5% and 117.1±3.6%, respectively. All the organic solvents except glycerol, iso-Propyl alcohol, acetonitrile and n-butanol could feed-back the activity of M372 I AK. Lysine inhibition function was relieved to some extent.3. The kinetic parameters Vmax of mutant G377 D, G377 F, G377 Y and G377 K AK were 1.93, 9.16, 6.55 and 7.22 times higher than that of WT AK, respectively. The n value of the mutation AK was 1.1, 1.0, 1.3 and 1.3, respectively. Indicating the positive cooperativity of AK decreased. Thus, chosing G377 F AK do the further study. The results of enzymetic characterization showed that the optimum reaction pH of G377 F was 9.0, which was higher than that of WT(pH 8.5). The optimum reaction temperature of G377 F was the same as that of WT(25?C). Under the optimum reaction temperature and pH, the half-life period of G377 F was 5.3h. K+, Ca2+, Mn2+, Cu2+ and Fe3+ in low concentration(0.2mM) could improve the activity of G377 F AK. All the organic solvents except glycerol, iso-Propyl alcohol, acetonitrile and n-butanol could decrease the activity of G377 F AK. Lysine in low concentration(0.2, 1mM) could improve the activity of G377 F AK, the relative enzymatic activity was 111.5±1.5% and 101.4±0.8%, respectively.4. The kinetic parameters Vmax of mutant V378 F, V378 D and V378 H AK were 1.93, 5.86 and 2.01 times higher than that of WT AK, respectively. The n value of the mutation AK was 1.0, 1.1 and 1.0, respectively. Indicating the positive cooperativity of AK decreased. Km value of mutations AK except V378 H AK had all decreased than that of WT AK. Thus, chosing V378 F AK to do the enzymatic characterization study. The results showed that the optimum reaction pH of V378 F was 8.0 and the optimum reaction temperature of V378 F was 28?C. Under the optimum reaction temperature and pH, the half-life period of V378 F AK increased from 2.7h(WT) to 4.4h. K+, Zn2+, Mn2+, Cu2+ and Fe3+ in low concentration(0.2mM) could improve the activity of V378 F AK. All the organic solvents except acetonitrile and n-butanol could decrease the activity of V378 F AK. Lysine in low concentration(0.2, 1mM) could improve the activity of V378 F AK, the relative enzymatic activity was 121.5±1.6% and 100.4±1.0%, respectively. However, when the inhibition differences between in the combination of threonine+lysine and lysine+methionine was in low concentration(0.2mM), the relative enzymatic activity was 117.4±1.3% and 122.0±2.0%, respectively. Compared with WT, it showed that lysine inhibition function was relieved to some extent.