Molecular basis for substrate selectivity and specificity by an LPS biosynthetic enzyme

Diversity in the polysaccharide component of lipopolysaccharide (LPS) contributes to the persistence and pathogenesis of Gram-negative bacteria. The Nudix hydrolase GDP-mannose mannosyl hydrolase (Gmm) contributes to this diversity by regulating the concentration of mannose in LPS biosynthetic pathways. Here, we present seven high-resolution crystal structures of Gmm from the enteropathogenic E. coli strain O128: the structure of the apo enzyme, the cocrystal structure of Gmm bound to the product Mg²⁺ - GDP, two cocrystal structures of precatalytic and turnover complexes of Gmm-Ca ²⁺ - GDP-α-D-mannose, and three cocrystal structures of an inactive mutant (His-124 → Leu) Gmm bound to substrates GDP-α-D-mannose, GDP-α-D-glucose, and GDP-β-L-fucose. These crystal structures help explain the molecular basis for substrate specificity and promiscuity and provide a structural framework for reconciling previously determined kinetic parameters. Unexpectedly, these structures reveal concerted changes in the enzyme structure that result in the formation of a catalytically competent active site only in the presence of the substrate/product. These structural views of the enzyme may provide a rationale for the design of inhibitors that target the biosynthesis of LPS by pathogenic bacteria.