Bacterium Bacillus Subtilis. Preparation from Bacillus Subtilis B-3157 of 2H-Labeled Purine Ribonucleoside Inosine

Abstract

This paper deals with studying biosynthetic pathways of ²H-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 ²H-labeled growth substrates. Isolation of ²H-labeled inosine from LC was performed by adsorption/desorption on activated carbon with following extraction by 0.3 M ammonium–formate 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 ammonium–formate buffer and 0.045 M NH₄Cl. The investigation of deuterium incorporation into the inosine molecule by FAB method demonstrated incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment – 65.5 atom% ²H) with 3 deuterium atoms being included into the ribose and 2 deuterium atoms – into the hypoxanthine residue of the molecule. Three non-exchangeable deuterium atoms were incorporated into the ribose residue owing to the preservation in this bacterium the minor pathways of de novo glucose biosynthesis in ²H₂O-medium. These non-exchangeable deuterium atoms in the ribose residue were originated from HMP shunt reactions, while two other deuterium atoms at C2,C8-positions in the hypoxanthine residue were synthesized from [²H]amino acids, primarily glutamine and glycine, that originated from deuterated hydrolysate. A glycoside proton at b-N₉-glycosidic bond could be replaced with deuterium via the reaction of ??₂ elimination at the stage of ribulose-5-monophosphate formation from 3-keto-6-phosphogluconic acid with subsequent proton (deuteron) attachment at the ?1-position of ribulose-5-monophosphate. Two other protons at C2(C3) and C4 positions in ribose residue could be replaced with deuterium via further enzimatic isomerization of ribulose-5-monophosphate into ribose-5-monophosphate.

Key words: ²H-labeled inosine, biosynthesis, biosynthetic pathways, heavy water, Bacillus subtilis