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

Abstract

SummaryThe 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.

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

“…Therefore, further biochemical and structural information on this pathway in additional species is important to optimize lead compounds for improved affinity and specificity through rational design. Recently, we characterized GAS GacA as a RmlD homolog catalyzing the last step in dTDP-L-rhamnose biosynthesis (van der Beek et al, 2015). Surprisingly, this GAS RmlD enzyme was confirmed to be a monomer as opposed to previously characterized RmlD dimers.…”

“…Most genes directly or indirectly involved in the GAC biosynthesis pathway are essential for GAS viability, including all four dTDP-L-rhamnose biosynthesis genes rmlABC and gacA (Le Breton et al, 2013;van der Beek et al, 2015;Zhu et al, 2017). In contrast and for unknown reasons, the dTDP-L-rhamnose biosynthesis genes are not essential in S. mutans under noncompetitive conditions.…”

“…In contrast, all GAS serotypes express a structurally invariant GAC (Lancefield, 1933). Similar to classical wall teichoic acids, rhamnose cell wall polysaccharides are critical for maintaining cell shape, bacterial physiology and virulence, but in-depth knowledge of their biosynthesis or host interactions at a molecular level is limited (van Sorge et al, 2014;van der Beek et al, 2015;Kovacs et al, 2017;Zhu et al, 2017). A better understanding of these mechanisms could aid the development of new classes of antibiotics, antibiotic adjuvants or vaccines (Sabharwal et al, 2006;van Sorge et al, 2014;St Michael et al, 2017).…”

“…In the first step of the pathway, RmlA, a glucose-1-phosphate thymidyltransferase, converts glucose-1-phosphate into dTDP-glucose (Blankenfeldt et al, 2000), which is subsequently oxidized and dehydrated to form dTDP-4-keto-6deoxy-D-glucose by the dTDP-D-glucose 4,6-dehydratase RmlB (Beis et al, 2003). RmlC catalyzes an unusual double epimerization reaction Dong et al, 2003b;2007), the product of which is finally reduced by RmlD, a dTDP-4-dehydrorhamnosereductase, to form dTDP-L-rhamnose (Blankenfeldt et al, 2002;van der Beek et al, 2015). The mechanisms of action as well as structural characteristics of the RmlABCD enzymes have been studied extensively (Blankenfeldt et al, 2000;Allard et al, 2001;Beis et al, 2003;Dong et al, 2003a;2003b;Kantardjieff et al, 2004;van der Beek et al, 2015).…”

“…RmlC catalyzes an unusual double epimerization reaction Dong et al, 2003b;2007), the product of which is finally reduced by RmlD, a dTDP-4-dehydrorhamnosereductase, to form dTDP-L-rhamnose (Blankenfeldt et al, 2002;van der Beek et al, 2015). The mechanisms of action as well as structural characteristics of the RmlABCD enzymes have been studied extensively (Blankenfeldt et al, 2000;Allard et al, 2001;Beis et al, 2003;Dong et al, 2003a;2003b;Kantardjieff et al, 2004;van der Beek et al, 2015). Indeed, crystal structures are available for all four Rml enzymes from different Gram-positive and Gram-negative bacterial species and show high structural conservation.…”