GacA is essential for Group A Streptococcus 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 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.
University Medical Center Utrecht, Medical Microbiology, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
Samantha L
SL
van der Beek
Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, 3124 Biosciences Research Building, College Park, MD 20742, USA.
Yoann
Y
Le Breton
Division of Molecular Microbiology, University of Dundee, School of Life Sciences, Dow Street, DD1 5EH, Dundee, UK.
Andrew T
AT
Ferenbach
Complex Carbohydrate Research Center, Department of Chemistry, The University of Georgia, 315 Riverbend Road, Athens, USA.
Robert N
RN
Chapman
Division of Molecular Microbiology, University of Dundee, School of Life Sciences, Dow Street, DD1 5EH, Dundee, UK.
Daan M F
DM
van Aalten
Division of Biological Chemistry and Drug Discovery, University of Dundee, School of Life Sciences, Dow Street, DD1 5EH, Dundee, UK.
Iva
I
Navratilova
Complex Carbohydrate Research Center, Department of Chemistry, The University of Georgia, 315 Riverbend Road, Athens, USA.
Geert-Jan
GJ
Boons
Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, 3124 Biosciences Research Building, College Park, MD 20742, USA.
Kevin S
KS
McIver
University Medical Center Utrecht, Medical Microbiology, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
Nina M
NM
van Sorge
Division of Molecular Microbiology, University of Dundee, School of Life Sciences, Dow Street, DD1 5EH, Dundee, UK.|Rutherford Appleton Laboratory, Research Complex at Harwell, OX11 0FA, Didcot, UK.
Helge C
HC
Dorfmueller
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002238:Carbohydrate Epimerases / Q000378:metabolism; D003001:Cloning, Molecular; D006094:Gram-Positive Bacteria / Q000201:enzymology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis; D017434:Protein Structure, Tertiary; D012210:Rhamnose / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D016415:Sequence Alignment; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics; D013942:Thymine Nucleotides / Q000096:biosynthesis / Q000378:metabolism
D001426:Bacterial Proteins; C091100:GacA protein, Bacteria; D009702:Nucleoside Diphosphate Sugars; D013942:Thymine Nucleotides; C119260:dTDP-4-ketorhamnose; C073293:thymidine diphosphate rhamnose; D002237:Carbohydrate Dehydrogenases; C107144:dTDP-4-dehydrorhamnose reductase; D002238:Carbohydrate Epimerases; D012210:Rhamnose