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.
Highlights
Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) derive from riboflavin (RF, vitamin B2) and act as cofactors of flavoproteins
The independently expressed riboflavin kinase (RFK) module of CaFADS retains the ability to catalyze the phosphorylation of RF and conserves the structural determinants that are responsible for strong inhibition by its RF substrate[18]
Our results lead us to conclude that the collective binding of adenine and flavin nucleotides induces important conformational changes in the CaFADS RFK module
Summary
Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) derive from riboflavin (RF, vitamin B2) and act as cofactors of flavoproteins. We focus on these two particular facts while characterizing the isolated RFK module of CaFADS (Δ(1–182)CaFADS) This truncated form of the enzyme has shown to perform the RFK activity with ligand binding profiles and strong substrate inhibition that are similar to those observed in the full-length bifunctional enzyme[18]. Isothermal titration calorimetry experiments outline the intricate ensemble of protein complexes to identify the most favorable pathways and to predict cooperativity between ligands These results are discussed in the framework of the crystal structures of the RFK module of CaFADS, both free and in complex with its products[14, 18], to evaluate the conformational changes that are produced during ligand binding and RFK catalysis
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