Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169

International Journal of Molecular Sciences

Weihong Min¹  Huiying Li¹  Hongmei Li¹  Chunlei Liu¹  Jingsheng Liu¹ 

[1] College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China;

关键词: Corynebacterium pekinense;    aspartate kinase;    characterization;    molecular docking;   

DOI  :  10.3390/ijms161226098

来源: mdpi

【 摘 要 】

Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni²⁺. Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter V_max of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with l-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production.

【 授权许可】

CC BY   
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