Mechanism of the feedback-inhibition resistance in aspartate kinase of Corynebacterium pekinense: from experiment to MD simulations

Abstract

Corynebacterium pekinense AK was successfully modified and two mutants A380C and T379N/A380C with high enzyme activity were constructed. The mechanism of their feedback-inhibition resistance was thoroughly studied from experiment to MD simulations.

Characterization of aspartokinase double mutants using a combination of experiments and simulations

“…The single mutants A380I, T379L, and A380 M and double mutant T379 N/A380C were obtained by inducing saturation mutations and high-throughput screening, and the enzyme activity was improved; however, enzymatic properties were not improved. Our previous studies confirmed that sites 379 and 380 are important sites affecting enzyme activity [ 12 , 13 ]. In this study, we used sites 379 and 380 as saturated mutation sites ( Fig.…”

Improving Microbial Cell Factory Performance by Engineering SAM Availability

“…44 The double mutant (T379N/A380C) AK from Corynebacterium pekinense showed 22.79 times higher activity than that of the wild-type one. 152 MTHFR (5,10-ethylenetetrahydrofolate reductase) catalyzes the reduction of 5,10-methylene-THF to 5-MTHF, which serves as an in vivo methyl donor. MTHFRs from mammalian and yeast are feedback inhibited by SAM, while those from plants are not.…”

“…52 As aforementioned, aspartate kinase (AK), which catalyzes the phosphorylation of aspartate, suffers from feedback inhibition and transcription repression by end products (such as L-threonine, isoleucine, or SAM) through allosteric effect. 152 AKs from different organisms have different inhibitory effectors and regulatory sites. For instance, aspartate kinase I (AKI, encoded by thrA) from E. coli is feedback inhibited by L-threonine and L-homoserine; this inhibition can be released by a site mutation (C1034T).…”

“…As aforementioned, aspartate kinase (AK), which catalyzes the phosphorylation of aspartate, suffers from feedback inhibition and transcription repression by end products (such as l -threonine, isoleucine, or SAM) through allosteric effect . AKs from different organisms have different inhibitory effectors and regulatory sites.…”

“…coli is feedback inhibited by l -threonine and l -homoserine; this inhibition can be released by a site mutation (C1034T) . The double mutant (T379N/A380C) AK from Corynebacterium pekinense showed 22.79 times higher activity than that of the wild-type one . MTHFR (5,10-ethylenetetrahydrofolate reductase) catalyzes the reduction of 5,10-methylene-THF to 5-MTHF, which serves as an in vivo methyl donor.…”