Relieving Feedback Inhibition And Characterization Of Enzymatic Properties For Aspartate Kinase From Corynebacterium Pekinense

Aspartate kinase（AK） is the first rate-limiting enzyme for amino acids of aspartate family in microbial fermentation. The feedback inhibition by end-product of aspartate pathway leading to low yield for amino acids of aspartate family,especially for methionine. We take CpAK（AK from Corynebacterium pekinenseas） as research object on the basis of a successful connection between CpAK gene and carrier pET- 28 a. There are three important amino acid residues associated with feedback inhibitation were identified by taking CgAK（AK from Corynebacterium glutamicum） as a reference. Site-saturation mutagenesis and high-throughput screening were implemented in order to relieve feedback inhibition and obtain high vitality CpAKs and the characterization were carried out as well. The results are as follows:1. The sequence similarity of amino acid between CpAK and CgAK is 99% through DNAMAN sequence alignment and they are both collaborative feedback inhibited by lysine and threonine, according to our previous studies, threonine plays a leading role.So we speculated that the regulating mechanism and the spatial structure of CpAK were similar with CgAK. So using multiple sequence alignment technology and bioinformatics, we identified three important residues associate with Thr501 with taking 3aaw（the structure of CgAK） as reference. The three residues are A297, D312 and Q316. After design of primers and site-saturation mutagenesis, putting plasmids of PET-28a-AK into e.coli（Escherichia coli） BL21 respectively. Then eight high vitality mutants were obtained named A297P、A297K、D312R、D312C、D312I、Q316R、Q316A、Q316N after high-throughput screening.2. Bacterium suspension was obtained after activating bacteria and induced expression.then ultrasonication and Ni²⁺–NTA column were used to prepare crude enzyme and refined solution of CpAK respectively. Native- PAGE, SDS-PAGE and Western blot proved CpAK has realized overexpression in e. coli BL21 and molecular weight of CpAK was about 130 kDa, while the molecular weight of α subunit and β subunit were about 47 kDa and 18 kDa respectively. According to the results we speculated that CpAK and CgAK were both hereotetramer.3. Substrate enzymatic dynamics analysis was implemented for WT AK（CpAK which have no mutation）, A297 P AK and A297 K AK. The results showed that Vmax of A297 P AK and A297 K AK increased by 8.95 times and 9.55 times, respectively, Km value of the mutations AK both improved than that of WT AK and n value of the mutations AK both decreased than that of WT AK. Thus, A297 K AK which has the most significantly Vmax change was chosen to go on the characterization study. The results are as follows: The optimum reaction temperature was 30 ℃that has increased compared with WT AK whose optimum temperature was 25 ℃; The optimum pH was still 8.0; And the stability of A297 K AK uder the optimal conditions extended from 3.5h to 5 h; Ni²⁺ and Mg²⁺ could improve the activity of A297 K AK with low concentration; Methanol and isopropanol also could improve the activity of A297 K AK with low concentration. The inhibition caused by Thr, Lys and the synergistic inhibition caused by Thr and Lys were successfully released under low concentration,which was consistent with research objective. Structure analysis showed that,after mutation, on the one hand, stereo-hindrance effect strengthened because of the long side chain of Lys297.On the other hand,R- nitrogen atom of Lys297 bonded with oxygen atom of Gln316 in α subunit and oxygen atom of Ile126 in β subunit because the distances were shortened to 2.32 ? and 3.29 ? respectively. Considering the above,Thr501 was blocked outside the binding pocket so that changes occurred in the whole structure of the enzyme.4. Substrate enzymatic dynamics analysis was implemented for WT AK, D312 R AK, D312 C AK and D312 I AK. The results showed that Vmax of D312 R AK,D312 C AK and D312 I AK increased by 5.02 times,7.79 times and 10.07 times, respectively, except for D312 C AK, Km value of other mutations AK both improved than that of WT AK and n value of the mutations AK all decreased than that of WT AK to varying degrees. Thus, D312 I AK hich has the most significantly Vmax change was chosen to go on the characterization study. The results are as follows: The optimum reaction temperature was 30 that has increased compared with ℃WT AK whose optimum temperature was 25 ℃;The optimum pH was still 8.0;And the half-life of D312 I AK uder the optimal conditions was still 3.5h; Mn²⁺ and Ni²⁺ could improve the activity of D312 I AK; acetonitrile could improve the activity of D312 I AK with low concentration to varing degrees. Combinations of different inhibitors showed no apparent activation to enzyme activity of D312 I AK. But the trends to inhibition overall were abated, which was consistent with research objective. Structure analysis showed that, after mutation, Ile312 had no relevance with Lys601 because its side chain could not bond with Ala224 through water molecule; At the same time. There was no interaction between Ile312 and neighboring amino acid due to the mutation, and then this change weakened the tension between D312 I AK and short peptide Met313-Val314-Leu315-Gln316 leading to threonine binding loosely. Above-mentioned two kinds of interrelation were broken may leading to changes in structure and resulted in relax state of AK which was adverse to collaborative feedback inhibition.5. Substrate enzymatic dynamics analysis was implemented for WT AK, Q316 R AK, Q316 A AK and Q316 N AK. The results showed that Vmax of Q316 R AK, Q316 A AK and Q316 N AK increased by 2.26 times, 3.13 times and 3.07 times, respectively, Km value of the mutations AK all improved than that of WT AK and n value of the mutations AK decreased than that of WT AK. Thus, Q316 A AK which has the most significantly Vmax change was chosen to go on the characterization study. The results are as follows: The optimum reaction temperature was 30 ℃that has increased compared with WT AK whose optimum temperature was 25 ℃;The optimum pH was 7.5 that has decreased compared with WT AK whose optimum pH was 8.0; the stability of Q316 A AK uder the optimal conditions extended from 3.5h to 4 h; Na+,Zn²⁺,Ni²⁺ and Mg²⁺ could improve the activity of Q316 A AK with low concentration; Na+,Zn²⁺,Ni²⁺ and Mg²⁺ could improve the activity of Q316 A AK with low concentration; the inhibition of ethanol to enzyme was decreased significantly but still exists; The synergistic feedback inhibition caused by different inhibitors were successfully released under low concentration, which was consistent with research objective. Structure analysis showed that, after mutation, side chain of Ala316 losed amide group leading to intermolecular forces between Ala316 and Thr501 disappeared with the distance between them increased to 5.55 A. And then the instable binding of Thr501 weakened collaborative feedback inhibition.