Expression and purification of the 5′-nucleotidase YitU from Bacillus species: its enzymatic properties and possible applications in biotechnology

Abstract

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.

Field evaluation of PGP Bacillus sp. strain D5 native to Crocus sativus, in traditional and non traditional areas, and mining of PGP genes from its genome

“…The heat and desiccation-resistant spores make it more potent biological control agents. The ease of stable formulation, GRAS (generally regarded as safe) status by the US food and drug administration are additional properties that make Bacillus most used plant growth promoting microbe 20 , 21 . A number of species of Bacillus such as B. pumilus, B. amyloliquefaciens, B. cereus, B. subtilis, B. licheniformis, B. megaterium, B. mycoides and B. Velezensis have been commercialized as bio-formulation 22 – 25 .…”

Microbial 5′-nucleotidases: their characteristics, roles in cellular metabolism, and possible practical applications

“…Microbial genomes can contain from very few to several dozen genes encoding HADSF phosphatases. Multiple genes that encode soluble HADSF phosphatases belonging to various HADSF subfamilies have been identified in the genomes of gram-negative (Proudfoot et al 2004 ; Kuznetsova et al, 2006 ) and gram-positive (Sarge et al 2015 ; Terakawa et al 2016 ; Zakataeva et al 2016 ; Yusupova et al 2020 ) bacteria, and their substrate specificities and the biochemical properties of their products have been studied. An important feature of these enzymes is that, unlike the classical “one enzyme for one substrate” model, most HADSF phosphatases possess remarkably broad and overlapping but nonidentical substrate profiles.…”

“…Recombinant YutF also exhibits phosphohydrolase activity against nucleotide precursors, ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate (Zakataeva et al 2016 ). The other B. subtilis HADSF phosphatases YcsE, YwtE, and YitU were shown to be involved in riboflavin (RF) biosynthesis, catalyzing the dephosphorylation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate (ARPP) in the de novo RF pathway and hydrolyzing FMN to RF (Sarge et al 2015 ; Yusupova et al 2020 ). Similar to all HADSF phosphatases, YcsE and YitU possess broad substrate specificity, demonstrating 5′-nucleotidase activity against various 5′-nucleotides (Terakawa et al 2016 ; Yusupova et al 2020 ).…”

“…The other B. subtilis HADSF phosphatases YcsE, YwtE, and YitU were shown to be involved in riboflavin (RF) biosynthesis, catalyzing the dephosphorylation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate (ARPP) in the de novo RF pathway and hydrolyzing FMN to RF (Sarge et al 2015 ; Yusupova et al 2020 ). Similar to all HADSF phosphatases, YcsE and YitU possess broad substrate specificity, demonstrating 5′-nucleotidase activity against various 5′-nucleotides (Terakawa et al 2016 ; Yusupova et al 2020 ). The other E. coli HADSF phosphatases YigB and YbjI preferentially use FMN as a substrate, but the paralogous protein SerB was shown to dephosphorylate phosphoserine (Kuznetsova et al 2006 ).…”

“…Moreover, several HADSF phosphatases were shown to directly participate in the histidine, serine, and FMN (RF) biosynthesis pathways (Rangarajan et al 2006 ; Kuznetsova et al 2006 ; Haase et al 2013 ). The B. subtilis HADSF phosphatases YcsE, YwtE, and YitU, which exhibit various affinities for ARPP and FMN, are involved in the synthesis and fine-tuning of intracellular pools of important flavins, such as RF, FMN, and FAD (Sarge et al 2015 ; Yusupova et al 2020 ). These data suggest the wider role of intracellular, microbial phosphatases/5′-nucleotidases in the HADSF in the regulation of cellular metabolism, which is not limited by the control of intracellular nucleotide pools but also extends to the control of pools of other key cellular metabolites.…”