Structure and function of sedoheptulose-7-phosphate isomerase, a critical enzyme for lipopolysaccharide biosynthesis and a target for antibiotic adjuvants.
The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of L-glycero-D-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism.
Department of Biochemistry and Biomedical Sciences, DeGroote School of Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada.
Patricia L
PL
Taylor
Kim M
KM
Blakely
Gladys P
GP
de Leon
John R
JR
Walker
Fiona
F
McArthur
Elena
E
Evdokimova
Kun
K
Zhang
Miguel A
MA
Valvano
Gerard D
GD
Wright
Murray S
MS
Junop
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D007700:Kinetics; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009675:Novobiocin / Q000494:pharmacology; D020836:Protein Structure, Quaternary; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013403:Sugar Phosphates / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001051:Apoenzymes; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D013403:Sugar Phosphates; D009675:Novobiocin; C020495:sedoheptulose 7-phosphate; D019998:Racemases and Epimerases; C445301:GmhA protein, E coli