Gram-negative bacteria (pathogens and nonpathogens) have a unique outer membrane and are more refractory to antibiotic therapy than most of their gram-positive counterparts (save mycobacteria). We previously provided genetic and biochemical evidence that defined the role for two postulated lipopolysaccharide (LPS) inner core biosynthetic genes (i.e., rfaC and rfaD). Our findings have relevance in the area of infection since gram-negative bacteria defective in LPS core synthesis have altered growth rate, virulence, and increased susceptibility to antibiotics. The E. coli K-12 rfaD gene product (i.e., ADP-L-glycero-D-mannoheptose 6-epimerase or epimerase) was previously crystallized in my laboratory. A diffraction resolution of 1.9 ? for epimerase crystals has been achieved. The 3 dimensional structure of ADP-L-glycero-D-mannoheptose 6-epimerase has been determined by the multi-wavelength anomalous dispersion (MAD) method at 3.0 A resolution. The anomalous scattering properties of selenium, incorporated into the protein as selenomethionine, were exploited in the phasing process. This is the largest protein structure (370 kDa) determined by the MAD phasing method. Sixty-five out of a total of 70 selenium atoms were located and used in the phasing process. Unexpectedly the enzyme crystallizes as a pentamer, with 2 pentamers in the asymmetric unit. Model building and crystallographic refinement to 2.0 A resolution is ongoing. We are also studying several chemically induced or site-directed mutations of the epimerase to further investigate the catalytic mechanism, structure and function of the enzyme. Recent mutational studies suggest that NAD binding is required for both catalytic activity and structural stability of epimerase. Helicobacter pylori plays a significant role in peptic ulcer disease; the mechanism of pathogenesis is not known at this time. We are using a mouse model for the study of H. pylori infection, disease mechanism and vaccine development. A CRADA between NIDDK and Diagnon Corporation is ongoing to identify and characterize novel targets for the development of antibiotics and protective vaccines directed against Helicobacter pylori. Two LPS core biosynthetic genes (rfaD and rfaE) from H. pylori have been cloned and the gene products purified. This is the first purification of the rfaE gene product from any gram-negative bacterium.
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