Wild-type and feedback-resistant phosphoribosyl pyrophosphate synthetases from Bacillus amyloliquefaciens: Purification, characterization, and application to increase purine nucleoside production

Bacillus strains are used for the industrial production of the purine nucleosides inosine and guanosine, which are raw materials for the synthesis of the flavor enhancers disodium inosinate and disodium guanylate. An important precursor of purine nucleosides is 5-phospho-α-d-ribosyl-1-pyrophosphate, which is synthesized by phosphoribosyl pyrophosphate synthetase (PRS, EC Class I PRSs are widespread in bacteria and mammals, are highly conserved among different organisms, and are negatively regulated by two end products of purine biosynthesis, adenosine 5′-diphosphate (ADP) and guanosine 5′-diphosphate (GDP). The D52H, N114S, and L129I mutations in the human PRS isozyme I (PRS1) have been reported to cause uric acid overproduction and gout due to allosteric deregulation and enzyme superactivity. In this study, to find feedback-resistant Bacillus amyloliquefaciens PRS, the influence of the D58H, N120S, and L135I mutations (corresponding to the D52H, N114S, and L129I mutations in PRS1, respectively) on PRS enzymatic properties has been studied. Recombinant histidine-tagged wild-type PRS and three mutant PRSs were expressed in Escherichia coli, purified, and characterized. The N120S and L135I mutations were found to release the enzyme from ADP and GDP inhibition and significantly increase its sensitivity to inorganic phosphate (P i) activation. In contrast, PRS with the D58H mutation exhibited nearly identical sensitivity to ADP and GDP as the wild-type protein and had a notably greater P i requirement for activation. The N120S and L135I mutations improved B. amyloliquefaciens and Bacillus subtilis purine nucleoside-producing strains. © 2011 Springer-Verlag.

Zakataeva N.P.1 , Romanenkov D.V.1 , Skripnikova V.S.1 , Vitushkina M.V.1 , Livshits V.A. 1, 2 , Kivero A.D.1 , Novikova A.E.1
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  • 1 Ajinomoto-Genetika Research Institute, 1-st Dorozhny Proezd, b.1-1, Moscow 117545, Russian Federation
  • 2 Department of General Pharmaceutical and Biomedical Technology, Peoples' Friendship University of Russia, Miklukho-Maklaya str., b.8, Moscow 117198, Russian Federation
Ключевые слова
Bacillus amyloliquefaciens; Bacillus subtilis; Feedback resistance; Phosphoribosyl pyrophosphate synthetase; Purine nucleoside-producing strains
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