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Identification and Characterization of Inhibitors of Bacterial Enoyl-Acyl Carrier Protein Reductase. Losee L. Ling, 2004.Bacterial enoyl-acyl carrier protein reductase (ENR) catalyzes an essential step in fatty acid biosynthesis . ENR is an attractive target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species . In addition, the bacterial ENR sequence and structural organization are distinctly different from those of mammalian fatty acid biosynthesis enzymes . High-throughput screening to identify inhibitors of Escherichia coli ENR yielded four structurally distinct classes of hits . Several members of one of these, the 2-(alkylthio)-4,6-diphenylpyridine-3-carbonitriles ("thiopyridines"), inhibited both purified ENR (50% inhibitory concentration [IC50] = 3 to 25 µM) and the growth of Staphylococcus aureus and Bacillus subtilis (MIC = 1 to 64 µg/ml) . The effect on cell growth is due in part to inhibition of fatty acid biosynthesis as judged by inhibition of incorporation of [14C]acetate into fatty acids and by the increased sensitivity of cells that underexpress an ENR-encoding gene (four- to eightfold MIC shift) . Synthesis of a variety of compounds in this chemical series revealed a correlation between IC50 and MIC, and the results provided initial structure-activity relationships . Preliminary structure-activity relationships, potency on purified ENR, and activity on bacterial cells indicate that members of the thiopyridine chemical series are effective fatty acid biosynthesis inhibitors suitable for further antibacterial development . Characterization and Heterologous Expression of the Oxalyl Coenzyme A Decarboxylase Gene from Bifidobacterium lactis. Federica Federici, 2004.Oxalyl coenzyme A (CoA) decarboxylase (Oxc) is a key enzyme in the catabolism of the highly toxic compound oxalate, catalyzing the decarboxylation of oxalyl-CoA to formyl-CoA . The gene encoding a novel oxalyl-CoA decarboxylase from Bifidobacterium lactis DSM 10140 (oxc) was identified and characterized . This strain, isolated from yogurt, showed the highest oxalate-degrading activity in a preliminary screening with 12 strains belonging to Bifidobacterium, an anaerobic intestinal bacterial group largely used in probiotic products . The oxc gene was isolated by probing a B . lactis genomic library with a probe obtained by amplification of the oxalyl-CoA decarboxylase gene from Oxalobacter formigenes, an anaerobic bacterium of the human intestinal microflora . The oxc DNA sequence analysis revealed an open reading frame of 1,773 bp encoding a deduced 590-amino-acid protein with a molecular mass of about 63 kDa . Analysis of amino acid sequence showed a significant homology (47%) with oxalyl-CoA decarboxylase of O . formigenes and a typical thiamine pyrophosphate-binding site that has been reported for several decarboxylase enzymes . Primer extension experiments with oxc performed by using RNA isolated from B . lactis identified the transcriptional start site 28 bp upstream of the ATG start codon, immediately adjacent to a presumed promoter region . The protein overexpressed in Escherichia coli cross-reacted with an anti-O . formigenes oxalyl-CoA decarboxylase antibody . Enzymatic activity, when evaluated by capillary electrophoresis analysis, demonstrated that the consumption substrate oxalyl-CoA was regulated by a product inhibition of the enzyme . These findings suggest a potential role for Bifidobacterium in the intestinal degradation of oxalate . Genetic and Biochemical Analysis of Phosphatase Activity of Escherichia coli NRII (NtrB) and Its Regulation by the PII Signal Transduction Protein. Augen A. Pioszak, 2003.Mutant forms of Escherichia coli NRII (NtrB) were isolated that retained wild-type NRII kinase activity but were defective in the PII-activated phosphatase activity of NRII . Mutant strains were selected as mimicking the phenotype of a strain (strain BK) that lacks both of the related PII and GlnK signal transduction proteins and thus has no mechanism for activation of the NRII phosphatase activity . The selection and screening procedure resulted in the isolation of numerous mutants that phenotypically resembled strain BK to various extents . Mutations mapped to the glnL (ntrB) gene encoding NRII and were obtained in all three domains of NRII . Two distinct regions of the C-terminal, ATP-binding domain were identified by clusters of mutations . One cluster, including the Y302N mutation, altered a lid that sits over the ATP-binding site of NRII . The other cluster, including the S227R mutation, defined a small surface on the "back" or opposite side of this domain . The S227R and Y302N proteins were purified, along with the A129T (NRII2302) protein, which has reduced phosphatase activity due to a mutation in the central domain of NRII, and the L16R protein, which has a mutation in the N-terminal domain of NRII . The S227R, Y302N, and L16R proteins were specifically defective in the PII-activated phosphatase activity of NRII . Wild-type NRII, Y302N, A129T, and L16R proteins bound to PII, while the S227R protein was defective in binding PII . This suggests that the PII-binding site maps to the "back" of the C-terminal domain and that mutation of the ATP-lid, central domain, and N-terminal domain altered functions necessary for the phosphatase activity after PII binding .
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