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 2.7.6.1). 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.

5'-Nucleotidases (EC 3.1.3.5) are enzymes that catalyze the hydrolytic dephosphorylation of 5′-ribonucleotides and 5′-deoxyribonucleotides to their corresponding nucleosides plus phosphate. In the present study, to search for new genes encoding 5′nucleotidases specific for purine nucleotides in industrially important Bacillus species, "shotgun" cloning and the direct selection of recombinant clones grown in purine nucleosides at inhibitory concentrations were performed in the Escherichia coli GS72 strain, which is sensitive to these compounds. As a result, orthologous yitU genes from Bacillus subtilis and Bacillus amyloliquefaciens, whose products belong to the ubiquitous haloacid dehalogenase superfamily (HADSF), were selected and found to have a high sequence similarity of 87%. B. subtilis YitU was produced in E. coli as an N-terminal hexahistidine-tagged protein, purified and biochemically characterized as a soluble 5′-nucleotidase with broad substrate specificity with respect to various deoxyribo-and ribonucleoside monophosphates: dAMP, GMP, dGMP, CMP, AMP, XMP, IMP and 5-aminoimidazole-4carboxamide-1-β-D-ribofuranosyl 5′-monophosphate (AICAR-P). However, the preferred substrate for recombinant YitU was shown to be flavin mononucleotide (FMN). B. subtilis and B. amyloliquefaciens yitU overexpression increased riboflavin (RF) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) accumulation and can be applied to breed highly performing RFand AICAR-producing strains.

No abstract