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 management of infection taking advantage of the observation that gram-negative bacteria defective in LPS core synthesis have altered growth rate, virulence, and increased susceptibility to antibiotics. My laboratory recently reported the crystallization and preliminary X-ray diffraction studies of the E. coli K-12 rfaD gene product (i.e., ADP-L-glycero-D- mannoheptose 6-epimerase or epimerase). Model building and crystallographic refinement to 2.0 angstroms resolution have now been completed. The structure of epimerase, in complex with NADP and the catalytic inhibitor ADP-glucose, has been determined at 2.0 angstroms resolution by multi-wavelength anomalous diffraction (MAD) methods. Epimerase is a homopentameric enzyme, which crystallizes with two pentamers in the asymmetric unit. The location of 70 crystallographically independent selenium sites was a key step in the structural determination. Each monomer subunit is arranged in two domains, a large N-terminal domain consisting of a seven-stranded Rossmann fold, associated with NADP binding and a smaller alpha/beta C- terminal domain involved in substrate binding. Based on our X-ray crystallographic studies, the catalytic mechanism of epimerase appears to be very similar to that proposed for the short-chain dehydrogenases. The presence of a Tyr XXX Lys motif in epimerase is generally a salient feature of the short-chain dehydrogenase superfamily and is ascribed a crucial catalytic function. We have constructed several chemically induced or site-directed epimerase mutant proteins (i.e., Y140C, Y140Q, K144I, K144R, S116A, G6S and G6A) and have begun to characterize the mutant proteins with respect to the catalytic mechanism and residues critical to NADP binding. Recent biochemical, analytical and structural studies of epimerase unequivocally indicate that epimerase is an NADP- dependent enzyme. However, inactive apo-epimerase (epimerase minus NADP) can be reconstituted with NAD with 50 percent enzymatic activity restored. The apparent Km values for NADP and NAD are 26 micromolar and 45 micromolar, respectively. We previously extended our investigation of the outer membrane to the gram-negative strain, Helicobacter pylori. H. pylori is the causative agent for gastritis, ulcer disease and some gastric cancers. The mechanism of H. pylori pathogenesis is not known at this time. We are using a mouse model for the study of H. pylori infection, detection methods, pathogenic mechanism(s) and vaccine development. We have developed a non-invasive, sensitive and species specific method for the antemortem detection method for investigating H. pylori infection of mice by PCR analysis of fecal pellets. DNA extracted from supernatant fractions of suspended fecal pellets was subjected to PCR analyses primed with H. pylori species specific primers. The primers target a genetic location on the 16S rRNA region that is specific to the H. pylori and amplify a 109 base pair fragment.A CRADA between NIDDK and Diagnon Corporation is ongoing with the aim 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, ADP-D-glycero-D-mannoheptose synthetase, from any gram-negative bacterium. The cloned LPS core- biosynthetic genes (rfaD and rfaE) of H. pylori genes are capable of complementing, respectively, E. coli or Salmonella typhimurium rfaD or rfaE mutants. - Permeability Barrier, Pathogenicity, Antibiotic Resistance, Gram-negative Bacteria, Escherichia coli, Helicobacter pylori, and uclers disease
