Expression and purification of the 5'-nucleotidase YitU from Bacillus species: its enzymatic properties and possible applications in biotechnology.
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-4-carboxamide-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 RF- and AICAR-producing strains.
['Yusupova|Yuliya R|YR|', 'Skripnikova|Victoria S|VS|', 'Kivero|Alexandr D|AD|', 'Zakataeva|Natalia P|NP|http://orcid.org/0000-0002-0976-9874']
D015720:5'-Nucleotidase D000620:Aminoimidazole Carboxamide D001407:Bacillus D000069977:Bacillus amyloliquefaciens D001412:Bacillus subtilis D001426:Bacterial Proteins D003001:Cloning, Molecular D004926:Escherichia coli D015870:Gene Expression D007700:Kinetics D011685:Purine Nucleotides D011994:Recombinant Proteins D012256:Riboflavin D012263:Ribonucleosides D013379:Substrate Specificity
Bacillus subtilis Bacillus amyloliquefaciens 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) producers 5′-Nucleotidases (EC 3.1.3.5) Riboflavin producers