Structure Reform Of Aspartate Kinase From Corynebacterium Pekinense

Aspartate kinase (AK) is an important key enzyme in the biosynthetic pathway ofaspartate-derived amino acids. Since the activity of AK is feedback inhibited by metabolite,aspartate-derived amino acids can’t be synthesized smoothly. To obtain a high yield ofaspartate-derived amino acids, AKs with high activity and without feedback inhibition arenecessary. This study aimed to modify AK gene from Corynebacterium pekinense on the basisof a successful recombination of pET-28a and this gene. Mutants with high activity wereconstructed by site-directed mutation combined with high throughput screening (HTS) and thecharacterization were studied as well. Researches and the results are as follows:1. Sequence alignment showed that AK gene from Corynebacterium pekinense has ahomology of more than98%with that from Corynebacterium glutamicum. Thus, AK fromCorynebacterium pekinense were studied in the reference of crystal structure of AK fromCorynebacterium glutamicum. By comparing the difference of crystal structures with or withoutinhibitors, we found that D292and R169are critical residues in the inhibition process inducedby threonine and lysine binding. So alteration of D292and R169could release the feedbackinhibition induced by threonine and lysine. The saturated mutant library of D292and R169wereconstructed. Then HTS was used to select mutation strains with high activity and D292Y, D292P,D292E, D292H, R169Y, R169D, R169P and R169H were obtained. AKs from those mutantsand WT were expressed in Escherichia coli BL21and were purified by Ni2+–NTA column afterdisrupting by sonication. The result of10%SDS-PAGE and Western blot of purified AKsshowed that AKs’ molecular weight was~48kDa.2. The kinetic parameters Vmax of mutant D292Y, D292P, D292E and D292H AK were8.11,3.87,3.23and2.32times higher, respectively, than that of WT AK. Km value of themutations AK except for D292Y AK had all improved in different degree than that of WT AK; nvalue of the mutations AK except for D292Y AK had all decreased in different degree than thatof WT AK. Thus, D292Y AK which had the biggest Vmax change was chosen to do thecharacterization study. The results of D292Y AK showed that the optimal temperature and pHwere30℃and8.5, both of which had increased comparing with those of WT AK; The half-lifeof D292Y AK was2.5h under optimal conditions and dropped by44%than that of WT AK; K+and Mg2+could improve the activity of D292Y AK under their optimal concentration; All theorganic solvents(methanol, ethanol, iso-propyl alcohol,acetonitrile, dimethyl sulfoxide, glycerol)except n-butanol used in this study could decrease the activity of D292Y AK;Inhibition on D292Y AK induced by threonine was disturbed; Both threonine and methioninecould improve the activity of D292Y AK under their optimal concentration.3. The kinetic parameters Vmax of mutant R169Y, R169D, R169P and R169H AK were4.71,2.57,2.25and2.13times higher, respectively, than that of WT AK. Km value of themutations AK had all improved in different degree than that of WT AK; n value of the mutationsAK had all decreased in different degree than that of WT AK. Thus, R169Y AK which had thebiggest Vmax change was chosen to do the characterization study. The results showed that theoptimal temperature and pH were35℃and9.0, both of which had increased comparing withthose of WT AK; The half-life of R169Y AK was5.5h under optimal conditions and improvedby22%than that of WT AK; Zn2+and Ni2+could improve the activity of R169Y AK when theirconcentration were5mM; All the organic solvents(methanol, iso-propyl alcohol, n-butanol,acetonitrile, dimethyl sulfoxide, glycerol)except ethanol used in this study could decrease theactivity of R169Y AK; Inhibition on R169Y AK induced by lysine was disturbed; Both lysineand methionine could improve the activity of R169Y AK under their optimal concentration.4. Mutant D292YR169Y was constructed and recombinant from D292Y was used astemplate. The kinetic parameters Vmax of mutant D292YR169Y AK was4.9times higher thanthat of WT AK. Characterization study showed that the optimal temperature and pH were35℃and9.0, both of which had increased comparing with those of WT AK; The half-life ofD292YR169Y AK was3.5h under optimal conditions and decreased by22%than that of WTAK; K+could improve the activity of D292YR169Y AK when its concentration was low; All theorganic solvents (methanol, ethanol, iso-propyl alcohol, n-butanol, acetonitrile, dimethylsulfoxide, glycerol)used in this study could decrease the activity of D292YR169Y AK; Tocorrespond with our study was that the feedback inhibition induced by threonine and lysine wasdisturbed in some degree. Threonine, lysine and methionine could improve the activity ofD292YR169Y AK when they were in optimal concentration.