TY - JOUR
T1 - GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP-4-dehydrorhamnose reductases (RmlD)
AU - van der Beek, Samantha L.
AU - le Breton, Yoann
AU - Ferenbach, Andrew T.
AU - Chapman, Robert N.
AU - van Aalten, Daan M. F.
AU - Navratilova, Iva
AU - Boons, Geert-Jan
AU - Mciver, Kevin S.
AU - van Sorge, Nina M.
AU - Dorfmueller, Helge C.
PY - 2015/12/1
Y1 - 2015/12/1
N2 - 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.
AB - 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.
UR - https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84983110940&origin=inward
UR - https://www.ncbi.nlm.nih.gov/pubmed/26278404
U2 - https://doi.org/10.1111/mmi.13169
DO - https://doi.org/10.1111/mmi.13169
M3 - Article
C2 - 26278404
SN - 0950-382X
VL - 98
SP - 946
EP - 962
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 5
ER -