Studying the Biosynthesis of 2H-labeled purine Ribonucleoside Inosine by a Chemoheterotrophic Bacterium Bacillus subtilis B-3157

Abstract

We studied the biosynthesis of 2 H-labeled purine ribonucleoside inosine excreted into liquid microbial culture (LC) by Gram-positive chemoheterotrophic bacterium Bacillus subtilis B-3157 while growing of this bacterium on heavy water (HW) medium with 2% (v/v) hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of 2 H-labeled growth substrates. Isolation of 2 H-labeled inosine from the LC was performed by adsorption/desorption on activated carbon with following extraction by 0,3 M ammoniumformate buffer (pH = 8,9), crystallization in 80% (v/v) EtOH, and ion exchange chromatography (IEC) on a column with AG50WX 4 cation exchange resin equilibrated with 0,3 M ammoniumformate buffer and 0,045 M NH 4 Cl. The investigation of deuterium incorporation into the inosine molecule by FAB method demonstrated the incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment -65,5 atom% 2 H) with 3 deuterium atoms included into the ribose and 2 deuterium atoms -into the hypoxanthine residue of the molecule. Three nonexchangeable deuterium atoms were incorporated into the ribose residue owing to reactions of enzymatic izomerization of glucose in 2 H 2 O-medium. These non-exchangeable deuterium atoms in the ribose residue were originated from glycolysis, associated with the Embden-Meyerhof pathway, while two other deuterium atoms at C2,C8-positions in the hypoxanthine residue were synthesized from [ 2 H]amino acids that originated from the deuterated hydrolysate of the methylotrophic bacterium Brevibacterium methylicum B-5662. However, the effect of auxotrophy of this strain in tyrosine, histidine, adenine and uracilб presupposes the branched metabolic pathways, different those indicated above.