Abstract: Aspartokinase（AK）is a key enzyme involved in the lysine biosynthesis,but its activity is synergistically inhibited by end-products such as lysine and threonine. This study focuses on finding new mutation to relieve the synergistic inhibition and unveiling this molecular mechanism form protein structure analysis. The amino acid sequences of AK from high lysine-producing strain Corynebacterium glutamicum ZL5 and wild-type（WT）C. glutamicum ATCC 13032 were aligned,and it was shown that G359D mutation was detected in aspartokinase from C. glutamicum ZL5. To discovery the biological function of this mutation,these WT and G359D Aks were expressed in Escherichia coli and were purified by the affinity chromatography;then,the purified recombined proteins were used for enzymatic activity detection when added the inhibitors lysine and threonine. The G359D protein displayed high resistance against the synergistic inhibition of lysine and threonine. The enzymatic activity of G359D remained at 76.94%±1.61% when the concentration of lysine and threonine reached 10 mmol/L,but the WT protein only was in 4.38%±1.28%. The similar result was also verified by the reverse mutation of G359D in the genome of C. glutamicum ZL5 producing high lysine yield,and the lysine yield decreased by 15.57%. From the further homology modeling and protein structure analysis,the G359D still interacted with lysine and threonine,but it enabled to relieve the allosteric effect of lysine,resulting from that the Arg¹⁵¹and Glu⁷⁴ in the catalytic active site of G359D could not form the ionic bond,thus allow the substrate into the active site. The aspartokinases G359D enabled to relieve the synergistic inhibition of lysine and threonine by blocking the allosteric effect of lysine.

Key words: Corynebacterium glutamicum, aspartokinase, lysine, threonine, synergistic inhibition