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

María Sebastián,Adrián Velázquez-Campoy,Milagros Medina

doi:10.1080/14756366.2018.1461857

Abstract

Emergence of multidrug-resistant bacteria forces us to explore new therapeutic strategies, and proteins involved in key metabolic pathways are promising anti-bacterial targets. Bifunctional flavin-adenine dinucleotide (FAD) synthetases (FADS) are prokaryotic enzymes that synthesise the flavin mononucleotide (FMN) and FAD cofactors. The FADS from the human pathogen Streptococcus pneumoniae (SpnFADS)–causative agent of pneumonia in humans − shows relevant catalytic dissimilarities compared to other FADSs. Here, by integrating thermodynamic and kinetic data, we present a global description of the riboflavin kinase activity of SpnFADS, as well as of the inhibition mechanisms regulating this activity. Our data shed light on biophysical determinants that modulate species-specific conformational changes leading to catalytically competent conformations, as well as binding rates and affinities of substrates versus products. This knowledge paves the way for the development of tools − that taking advantage of the regulatory dissimilarities during FMN biosynthesis in different species − might be used in the discovery of specific anti-pneumococcal drugs.

Highlights

Streptococcus pneumoniae is the causative agent of human pneumonia disease[1], meningitis and bacteremia in children and adults
flavin mononucleotide (FMN) and adenosine 50-diphosphate (ADP) bind to the free enzyme as well as to the SpnFADS:adenosine 50-triphosphate (ATP) and SpnFADS:RF complexes, respectively
Here we present a complete description of the riboflavin kinase activity (RFK) catalytic cycle of SpnFADS, integrating thermodynamic and kinetic data, both in the pre-steady and in the steady-state, obtained using different biophysical and biochemical tools

Summary

Introduction

Streptococcus pneumoniae is the causative agent of human pneumonia disease[1], meningitis and bacteremia in children and adults. When comparing SpnFADS with the member of the family so far best characterised—which is that from the organism Corynebacterium ammoniagenes (CaFADS)—it presents a very similar structure, with little differences in the position of some key loops. These two proteins only share the 26% of sequence homology[6]. Despite the overall structural similitude among prokaryotic FADSs6,12,18, SpnFADS shows three main distinctive functional behaviors; (i) it mainly stabilises monomers— which are the functional form6—or traces of dimers, during catalysis; (ii) its FMNAT activity requires reduced FMN as a substrate; and (iii) its RFK activity is not regulated by the RF substrate[6]

Methods

Results

Conclusion