Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis

Acta Naturae

article

Tatiana A. Shcherbakova¹  Semen M. Baldin¹  Mikhail S. Shumkov²  Irina V. Gushchina³  Dmitry K. Nilov¹  Vytas K. Švedas⁴ 

[1]Lomonosov Moscow State University, Belozersky Institute of Physicochemical Biology

[2]Federal Research Centre «Fundamentals of Biotechnology», Russian Academy of Sciences

[3]Lomonosov Moscow State University

[4]Lomonosov Moscow State University, Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University

关键词: transketolase;    thiamine diphosphate;    xylulose 5-phosphate;    ribose 5-phosphate;    mycobacteria;   

DOI  :  10.32607/actanaturae.11713

学科分类：生物技术

来源: Moskovskii Gosudarstvennyi Universitet im.M.V.Lomonosova/M.V.Lomonosov Moscow State University

【 摘 要 】

Transketolase, an enzyme of the pentose phosphate pathway, plays an important role in the functioning of mycobacteria. Using plasmid pET-19b carrying the Rv1449c gene of transketolase from Mycobacterium tuberculosis and an additional histidine tag, we isolated and purified recombinant transketolase and determined the conditions for obtaining the apoform of the protein. The Michaelis constants were evaluated for the thiamine diphosphate cofactor in the presence of magnesium and calcium ions. We found that the affinity of mycobacterial transketolase for thiamine diphosphate is by three orders of magnitude lower than that of the human enzyme. Analysis of the structural organization of the active centers of homologous enzymes showed that this difference is due to a replacement of lysine residues by less polar amino acid residues.

【 授权许可】

Unknown