Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes

Abstract

Enzymes known as bifunctional and bimodular prokaryotic type-I FAD synthetase (FADS) exhibit ATP:riboflavin kinase (RFK) and FMN:ATP adenylyltransferase (FMNAT) activities in their C-terminal and N-terminal modules, respectively, and produce flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These act as cofactors of a plethora of flavoproteins in all organisms. Therefore, regulation of their production maintains the cellular flavoproteome homeostasis. Here, we focus on regulation of the FMN synthesis in Corynebacterium ammoniagenes (Ca) by the inhibition of its RFK activity by substrates and products of the reaction. We use a truncated CaFADS variant consisting in the isolated C-terminal RFK module, whose RFK activity is similar to that of the full-length enzyme. Inhibition of the RFK activity by the RF substrate is independent of the FMNAT module, and FMN production, in addition to being inhibited by an excess of RF, is also inhibited by both of the reaction products. Pre-steady-state kinetic and thermodynamic studies reveal key aspects to the substrates induced fit to produce the catalytically competent complex. Among them, the role of Mg2+ in the concerted allocation of substrates for catalysis and the ensemble of non-competent complexes that contribute to the regulated inhibition of the RFK activity are particularly relevant.

The RFK catalytic cycle of the pathogen Streptococcus pneumoniae shows species-specific features in prokaryotic FMN synthesis

“…Reaction samples contained different concentrations of RF (0.5–30 µM) and ATP (10–500 µM), as previously described 21 , 22 . The inhibitory effect of the products of the reaction was analysed as previously described 23 . In short, the Spn FADS RFK activity was determined at increasing concentrations of FMN, varying the ATP concentration and keeping the RF constant (when studying the inhibitory effect of FMN), and at increasing concentrations of adenosine 5′-diphosphate (ADP), varying the RF concentration and keeping the ATP fixed (when studying the inhibitory effect of ADP).…”

“…Kinetic experiments in the pre-steady state were registered as previously described 23 , using stopped-flow spectroscopy on an Applied Photophysics SX17. MV spectrophotometer, using the Xscan software (Applied Photophysics Ltd., Leatherhead, UK).…”

“…MV spectrophotometer, using the Xscan software (Applied Photophysics Ltd., Leatherhead, UK). Fast kinetic measurements were carried out as previously described 23 , at 25 °C in PIPES 20 mM pH 7.0, 0.8 mM MgCl 2 . About 0.2 µM Spn FADS was mixed with reaction samples that contained increasing concentrations of the flavin ligands (FLV, herein indicating RF or FMN), in the presence and in the absence of ADP or ATP (herein referred as ANP).…”

“…Experiments were performed in triplicate. The flavin photobleaching was analysed as previously described 23 (not shown).…”

“…We performed isothermal titration calorimetry (ITC) assays to elucidate both the ligand binding order and the thermodynamic inhibition produced by the binding of the FLV and ANP ligands. Titrations were performed on an AutoITC200 (MicroCal, Malvern, UK) thermostated at 20 °C, as previously described 23 . Approximately 25 µM Spn FADS contained in a 200 µL-cell was titrated with solutions of 180 µM RF, 250 µM FMN, or 350 µM ATP or ADP.…”