Abstract
Sedoheptulose‐7‐phosphate isomerase, GmhA, is the first enzyme in the biosynthesis of nucleotide‐activated‐glycero‐manno‐heptoses and an attractive, yet underexploited, target for development of broad‐spectrum antibiotics. We demonstrated that GmhA homologs in Neisseria gonorrhoeae and N. meningitidis (hereafter called GmhAGC and GmhANM, respectively) were interchangeable proteins essential for lipooligosaccharide (LOS) synthesis, and their depletion had adverse effects on neisserial viability. In contrast, the Escherichia coli ortholog failed to complement GmhAGC depletion. Furthermore, we showed that GmhAGC is a cytoplasmic enzyme with induced expression at mid‐logarithmic phase, upon iron deprivation and anaerobiosis, and conserved in contemporary gonococcal clinical isolates including the 2016 WHO reference strains. The untagged GmhAGC crystallized as a tetramer in the closed conformation with four zinc ions in the active site, supporting that this is most likely the catalytically active conformation of the enzyme. Finally, site‐directed mutagenesis studies showed that the active site residues E65 and H183 were important for LOS synthesis but not for GmhAGC function in bacterial viability. Our studies bring insights into the importance and mechanism of action of GmhA and may ultimately facilitate targeting the enzyme with small molecule inhibitors.
Highlights
The World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) emphasized an urgent need for the development of antimicrobials with novel modes of action against antibiotic-resistant threats with severe consequences for public health, including infections caused by drug-resistant Neisseria gonorrhoeae (Centers for Disease Control and Prevention, 2013a, 2013b; World Health Organization, 2012, 2015)
To meet the needs raised by WHO and CDC, our laboratory focuses on identification and validation of new molecular targets for the development of gonorrhea treatments (Bonventre, Zielke, Korotkov, & Sikora, 2016; Zielke, Wierzbicki, Baarda, & Sikora, 2015; Zielke, Wierzbicki, Weber, Gafken, & Sikora, 2014; Zielke et al, 2016)
GmhA is conserved in many Gram-negative and some Gram-positive bacteria and is responsible for catalyzing isomerization of the D-sedoheptulose 7-phosphate into D-glycero-α,β-D- manno-heptose-7 -p hosphate (Eidels & Osborn, 1974), which is the first and common step for parallel biosynthetic pathways leading to generation of GDP-D-glycero-α-D-manno-heptose (D,D-heptose) and ADP-L-glycero-β-D-manno-heptose [L,D-heptose; reviewed in Ref: (Valvano et al, 2002)]
Summary
The World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) emphasized an urgent need for the development of antimicrobials with novel modes of action against antibiotic-resistant threats with severe consequences for public health, including infections caused by drug-resistant Neisseria gonorrhoeae (Centers for Disease Control and Prevention, 2013a, 2013b; World Health Organization, 2012, 2015). Nucleotide-activated-glycero- manno-heptoses, while absent in eukaryotic cells, are widely present in bacteria and are crucial components of the lipopolysaccharides (LPS), lipooligosaccharides (LOS), capsules, O-antigens, and glycan moieties of bacterial cell surface (S-layer) glycoproteins (Valvano et al, 2002). One such potential drug target of significant interest is sedoheptulose-7-phosphate isomerase, GmhA, annotated previously as TfrA (Havekes, Lugtenberg, & Hoekstra, 1976) and LpcA (Brooke & Valvano, 1996a). We performed characterization of GmhAGC at the molecular, functional, and structural levels to facilitate the future targeting of this enzyme with small molecule inhibitors
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