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

Mol Microbiol. 2015 Dec;98(5):946-62. doi: 10.1111/mmi.13169. Epub 2015 Oct 1.


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 Streptococcus (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 Salmonella 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 gacA in a S. mutans rmlD 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 gacA 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Carbohydrate Dehydrogenases / chemistry
  • Carbohydrate Dehydrogenases / genetics*
  • Carbohydrate Dehydrogenases / metabolism*
  • Carbohydrate Epimerases / metabolism
  • Cloning, Molecular
  • Gram-Positive Bacteria / enzymology
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Models, Molecular
  • Mutation
  • Nucleoside Diphosphate Sugars / biosynthesis
  • Protein Structure, Tertiary
  • Rhamnose / analogs & derivatives
  • Rhamnose / biosynthesis
  • Rhamnose / metabolism
  • Sequence Alignment
  • Streptococcus pyogenes / enzymology*
  • Streptococcus pyogenes / genetics
  • Thymine Nucleotides / biosynthesis
  • Thymine Nucleotides / metabolism


  • Bacterial Proteins
  • GacA protein, Bacteria
  • Nucleoside Diphosphate Sugars
  • Thymine Nucleotides
  • dTDP-4-ketorhamnose
  • thymidine diphosphate rhamnose
  • Carbohydrate Dehydrogenases
  • dTDP-4-dehydrorhamnose reductase
  • Carbohydrate Epimerases
  • Rhamnose