GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD). Issue 5 (1st October 2015)

• Record Type: Journal Article
• Authors: van der Beek, Samantha L.; Le Breton, Yoann; Ferenbach, Andrew T.; Chapman, Robert N.; van Aalten, Daan M. F.; Navratilova, Iva; Boons, Geert‐Jan; McIver, Kevin S.; van Sorge, Nina M.; Dorfmueller, Helge C.
• Is Part Of: Molecular microbiology. Volume 98:Issue 5(2015)
• Page Start: 946
• Page End: 962
• Publication Date: 2015-10-01
• Subjects: Molecular microbiology -- Periodicals; 572.829
• DOI: 10.1111/mmi.13169
• Languages: English
• ISSNs: 0950-382X

Abstract

Summary: The sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A S treptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium S almonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gac A in a S . mutans rml D knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gac A as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis. Abstract : L‐rhamnose is an important constituent of many bacterial cell walls and is essential for virulence and even viability. We characterized through genetics, biochemistry and structural biology the enzyme GacA, which catalyzes the final step in dTDP‐rhamnose biosynthesis, from the Gram‐positive human pathogen Group A Streptococcus (GAS). GacA is essential for GAS and, in contrast to a previous report, is enzymatically active as a monomer due to critical substitutions at the putative dimerization interface.