Abstract
Prokaryotic bifunctional FAD synthetases (FADSs) catalyze the biosynthesis of FMN and FAD, whereas in eukaryotes two enzymes are required for the same purpose. FMN and FAD are key cofactors to maintain the flavoproteome homeostasis in all type of organisms. Here we shed light to the properties of the hitherto unstudied bacterial FADS from the human pathogen Streptococcus pneumoniae (SpnFADS). As other members of the family, SpnFADS catalyzes the three typical activities of prokaryotic FADSs: riboflavin kinase (RFK), ATP:FMN:adenylyltransferase (FMNAT), and FAD pyrophosphorylase (FADpp). However, several SpnFADS biophysical properties differ from those of other family members. In particular; i) the RFK activity is not inhibited by the riboflavin (RF) substrate, ii) the FMNAT and FADSpp activities require flavin substrates in the reduced state, iii) binding of adenine nucleotide ligands is required for the binding of flavinic substrates/products and iv) the monomer is the preferred state. Collectively, our results add interesting mechanistic differences among the few prokaryotic bifunctional FADSs already characterized, which might reflect the adaptation of the enzyme to relatively different environments. In a health point of view, differences among FADS family members provide us with a framework to design selective compounds targeting these enzymes for the treatment of diverse infectious diseases.
Highlights
Prokaryotic FAD synthetases (FADSs) are bifunctional and bimodular enzymes that first catalyze the synthesis of flavin mononucleotide (FMN) from riboflavin (RF, vitamin B2) through an ATP:riboflavin kinase activity (RFK, EC 2.7.1.26) and subsequently the adenylylation of FMN to produce flavin adenine dinucleotide (FAD) using an ATP:FMN:adenylyltransferase activity (FMNAT, EC 2.7.7.2)[1,2,3]
The final absorption spectrum of purified SpnFADS exhibits a single peak at 279 nm (Supplementary Fig. S2) and in contrast to CaFADS13, SpnFADS does not co-purify with FAD, suggesting a much lower affinity for the oxidized flavinic substrates/products
Structural comparisons[10, 13, 36] and ligand binding models (Supplementary Fig. S1, and Fig. 5A) suggested that SpnFADS contains all the features of prokaryotic FADSs contributing to substrates stabilization and catalysis
Summary
Prokaryotic FAD synthetases (FADSs) are bifunctional and bimodular enzymes that first catalyze the synthesis of flavin mononucleotide (FMN) from riboflavin (RF, vitamin B2) through an ATP:riboflavin kinase activity (RFK, EC 2.7.1.26) and subsequently the adenylylation of FMN to produce flavin adenine dinucleotide (FAD) using an ATP:FMN:adenylyltransferase activity (FMNAT, EC 2.7.7.2)[1,2,3]. The different structures together with the significant differences at the catalytic centers between the N-terminal modules of bifunctional FADSs and the mammalian FMNAT enzymes[10, 14] make prokaryotic FADSs attractive exploitable targets for the treatment of human infectious diseases[37] In this context, SpnFADS might be considered as a drug target for S. pneumoniae, a pathogen almost exclusive for humans, that is the leading cause of invasive bacterial pneumoniae disease in children, in the elderly and in immunodepressed patients[38,39,40]. Our data indicate that SpnFADS catalyzes the RFK, FMNAT and FADpp activities It differs from other family members in two major catalytic aspects: its RFK activity does not exhibit inhibition by the RF substrate and the FMNAT activity requires the flavin substrate in its reduced state for substrate binding and catalysis. Our data point to these bacterial enzymes as promising pharmacological targets because differ between themselves and from the human enzyme
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