GacA is essential for Group A S treptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

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.