Streptococcal dTDP-L-rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Samantha L van der Beek, Azul Zorzoli, Ebru Çanak, Robert N Chapman, Kieron Lucas, Benjamin H Meyer, Dimitrios Evangelopoulos, Luiz Pedro S de Carvalho, Geert-Jan Boons, Helge C Dorfmueller, Nina M van Sorge

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Biosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP-L-rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP-L-rhamnose biosynthesis through their action as dTDP-glucose-4,6-dehydratase and dTDP-4-keto-6-deoxyglucose-3,5-epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio-layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP-rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose-dependent streptococcal pathogens as well as M. tuberculosis with an IC 50 of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP-L-rhamnose formation in a concentration-dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP-L-rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria.

Original languageEnglish
Pages (from-to)951-964
Number of pages14
JournalMolecular Microbiology
Volume111
Issue number4
Early online date1 Jan 2019
DOIs
Publication statusPublished - Apr 2019

Keywords

  • Anti-Bacterial Agents/isolation & purification
  • Biosynthetic Pathways
  • Hydro-Lyases/genetics
  • Inhibitory Concentration 50
  • Nucleoside Diphosphate Sugars/biosynthesis
  • Racemases and Epimerases/genetics
  • Streptococcus/drug effects
  • Thymine Nucleotides/biosynthesis

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