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-Ca2+-GDP-alpha-d-mannose, and three cocrystal structures of an inactive mutant (His-124 --> Leu) Gmm bound to substrates GDP-alpha-d-mannose, GDP-alpha-d-glucose, and GDP-beta-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.
Zou
Yaozhong
Y
Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, Illinois 61801, USA.
Li
Chong
C
Brunzelle
Joseph S
JS
Nair
Satish K
SK
D019943:Amino Acid Substitution; D001051:Apoenzymes / Q000737:chemistry; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D006155:Guanosine Diphosphate Mannose / Q000096:biosynthesis* / Q000378:metabolism*; D006156:Guanosine Diphosphate Sugars / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry*; D008956:Models, Chemical; D011487:Protein Conformation; D013379:Substrate Specificity
D001051:Apoenzymes; D029968:Escherichia coli Proteins; D006156:Guanosine Diphosphate Sugars; D020035:Holoenzymes; D008070:Lipopolysaccharides; D006154:Guanosine Diphosphate Fucose; D006155:Guanosine Diphosphate Mannose; C100085:guanosine diphosphate glucose; D007930:Leucine