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Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis.

ABSTRACT: Phosphoribosyltransferases are the tools that allow the synthesis of nucleotide analogues using multi-enzymatic cascades. The recombinant adenine phosphoribosyltransferase (TthAPRT) and hypoxanthine phosphoribosyltransferase (TthHPRT) from Thermus thermophilus HB27 were expressed in E.coli strains and purified by chromatographic methods with yields of 10-13 mg per liter of culture. The activity dependence of TthAPRT and TthHPRT on different factors was investigated along with the substrate specificity towards different heterocyclic bases. The kinetic parameters for TthHPRT with natural substrates were determined. Two nucleotides were synthesized: 9-(?-D-ribofuranosyl)-2-chloroadenine 5'-monophosphate (2-?l-AMP) using TthAPRT and 1-(?-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate (Allop-MP) using Tth?PRT.

SUBMITTER: Fateev IV

PROVIDER: S-EPMC6317416 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis.

Fateev Ilja V IV Sinitsina Ekaterina V EV Bikanasova Aiguzel U AU Kostromina Maria A MA Tuzova Elena S ES Esipova Larisa V LV Muravyova Tatiana I TI Kayushin Alexei L AL Konstantinova Irina D ID Esipov Roman S RS

Beilstein journal of organic chemistry 20181221

Phosphoribosyltransferases are the tools that allow the synthesis of nucleotide analogues using multi-enzymatic cascades. The recombinant adenine phosphoribosyltransferase (TthAPRT) and hypoxanthine phosphoribosyltransferase (TthHPRT) from Thermus thermophilus HB27 were expressed in E.coli strains and purified by chromatographic methods with yields of 10-13 mg per liter of culture. The activity dependence of TthAPRT and TthHPRT on different factors was i ...[more]

PMID: 30643587

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Project description:BackgroundThermus thermophilus, an extremely thermophilic bacterium, has been widely recognized as a model organism for studying how microbes can survive and adapt under high temperature environment. However, the thermotolerant mechanisms and cellular metabolism still remains mostly unravelled. Thus, it is highly required to consider systems biological approaches where T. thermophilus metabolic network model can be employed together with high throughput experimental data for elucidating its physiological characteristics under such harsh conditions.ResultsWe reconstructed a genome-scale metabolic model of T. thermophilus, iTT548, the first ever large-scale network of a thermophilic bacterium, accounting for 548 unique genes, 796 reactions and 635 unique metabolites. Our initial comparative analysis of the model with Escherichia coli has revealed several distinctive metabolic reactions, mainly in amino acid metabolism and carotenoid biosynthesis, producing relevant compounds to retain the cellular membrane for withstanding high temperature. Constraints-based flux analysis was, then, applied to simulate the metabolic state in glucose minimal and amino acid rich media. Remarkably, resulting growth predictions were highly consistent with the experimental observations. The subsequent comparative flux analysis under different environmental conditions highlighted that the cells consumed branched chain amino acids preferably and utilized them directly in the relevant anabolic pathways for the fatty acid synthesis. Finally, gene essentiality study was also conducted via single gene deletion analysis, to identify the conditional essential genes in glucose minimal and complex media.ConclusionsThe reconstructed genome-scale metabolic model elucidates the phenotypes of T. thermophilus, thus allowing us to gain valuable insights into its cellular metabolism through in silico simulations. The information obtained from such analysis would not only shed light on the understanding of physiology of thermophiles but also helps us to devise metabolic engineering strategies to develop T. thermophilus as a thermostable microbial cell factory.

Dataset Information

Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis.

Publications

Thermophilic phosphoribosyltransferases <i>Thermus thermophilus</i> HB27 in nucleotide synthesis.

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