Preparation of Flavocoenzyme Isotopologues by Biotransformation of Purines

Abstract

Isotope-labeled flavins are crucial reporters for many biophysical studies of flavoproteins. A purine-deficient Escherichia coli strain engineered for expression of the ribAGH genes of Bacillus subtilis converts isotope-labeled purine supplements into the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, with yields up to 40%. The fermentation products can subsequently be converted into isotope-labeled riboflavin and the cognate flavocoenzymes, FMN and FAD, by in vitro biotransformation with better than 90% yield. Using this approach, more than 100 single or multiple (13)C-, (15)N-, (17)O-, and (18)O-labeled isotopologues of these cofactors and ligands become easily accessible, enabling advanced ligand-based spectroscopy of flavoproteins and lumazine receptor proteins at unprecedented resolution.

Long-Lived Hydrated FMN Radicals: EPR Characterization and Implications for Catalytic Variability in Flavoproteins

“…Samples were frozen and stored under liquid nitrogen unless otherwise noted. Selectively labeled FMN was synthesized by published procedures. , …”

“…Selectively labeled FMN was synthesized by published procedures. 17,18 X-Band EPR Spectroscopy. X-band EPR experiments were carried out using a Bruker EMX spectrometer equipped with a Bruker X-SHQ 4119HS-W1 X-band resonator and a Bruker ER 4131VT digital temperature control system.…”

Site-Selective Synthesis of 15N- and 13C-Enriched Flavin Mononucleotide Coenzyme Isotopologues

“…GTP and ribulose 5-phosphate are precursors for the biosynthesis of riboflavin through the sequence of steps catalyzed by GTP cyclohydrolase II, lumazine synthase, and riboflavin synthase . Recently, Fischer and co-workers reported an in vivo biotransformation method using an engineered bacterial strain to synthesize 15 N-, 13 C-, 17 O-, and 18 O-labeled flavins from isotopically labeled precursors . In another report, Kohen and co-workers reported an enzymatic synthesis of 3 H-, 15 N-, and 13 C-labeled flavins from commercially available isotope-labeled riboflavin precursors .…”

“…16 Site-specifically 15 O-labeled flavins from isotopically labeled precursors. 23 In another report, Kohen and co-workers reported an enzymatic synthesis of 3 H-, 15 N-, and 13 C-labeled flavins from commercially available isotope-labeled riboflavin precursors. 24 We now report a combination of chemical and biotransformation steps for labeling nitrogen and carbon sites in the pyrazine and pyrimidine rings of the isoalloxazine core in FMN.…”