Dataset Information

Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis.

ABSTRACT: 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.

SUBMITTER: Shcherbakova TA

PROVIDER: S-EPMC9307985 | biostudies-literature | 2022 Apr-Jun

REPOSITORIES: biostudies-literature

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Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis.

Shcherbakova T A TA Baldin S M SM Shumkov M S MS Gushchina I V IV Nilov D K DK Švedas V K VK

Acta naturae 20220401 2

Transketolase, an enzyme of the pentose phosphate pathway, plays an important role in the functioning of mycobacteria. Using plasmid pET-19b carrying the <i>Rv1449c</i> gene of transketolase from <i>Mycobacterium tuberculosis</i> 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 calc ...[more]

PMID: 35923567

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Project description:The Mycobacterium tuberculosis genome harbors nine toxin-antitoxin (TA) systems that are members of the mazEF family, unlike other prokaryotes, which have only one or two. Although the overall tertiary folds of MazF toxins are predicted to be similar, it is unclear how they recognize structurally different RNAs and antitoxins with divergent sequence specificity. Here, we have expressed and purified the individual components and complex of the MazEF6 TA system from M. tuberculosis. Size exclusion chromatography-multiangle light scattering (SEC-MALS) was performed to determine the oligomerization status of the toxin, antitoxin, and the complex in different stoichiometric ratios. The relative stabilities of the proteins were determined by nano-differential scanning fluorimetry (nano-DSF). Microscale thermophoresis (MST) and yeast surface display (YSD) were performed to measure the relative affinities between the cognate toxin-antitoxin partners. The interaction between MazEF6 complexes and cognate promoter DNA was also studied using MST. Analysis of paired-end RNA sequencing data revealed that the overexpression of MazF6 resulted in differential expression of 323 transcripts in M. tuberculosis. Network analysis was performed to identify the nodes from the top-response network. The analysis of mRNA protection ratios resulted in identification of putative MazF6 cleavage site in its native host, M. tuberculosis. IMPORTANCE M. tuberculosis harbors a large number of type II toxin-antitoxin (TA) systems, the exact roles for most of which are unclear. Prior studies have reported that overexpression of several of these type II toxins inhibits bacterial growth and contributes to the formation of drug-tolerant populations in vitro. To obtain insights into M. tuberculosis MazEF6 type II TA system function, we determined stability, oligomeric states, and binding affinities of cognate partners with each other and with their promoter operator DNA. Using RNA-seq data obtained from M. tuberculosis overexpression strains, we have identified putative MazF6 cleavage sites and targets in its native, cellular context.

Dataset Information

Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis.

Publications

Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis.

Similar Datasets

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