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Staphylococcus aureus Mevalonate Kinase: Isolation and Characterization of an Enzyme of the Isoprenoid Biosynthetic Pathway. Natalya E. Voynova, 2004.It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate . Mevalonate kinase catalyzes a key reaction in this pathway . In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized . The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa . The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5 . The apparent Km values for R,S-mevalonate and ATP were 41 and 339 µM, respectively . There was substantial substrate inhibition at millimolar levels of mevalonate . The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized . These compounds were competitive inhibitors with respect to ATP; the Ki values were 46 and 45 µM for farnesyl diphosphate and its thio analog, respectively . Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S . aureus mevalonate kinase is much less sensitive to feedback inhibition (Ki difference, 3 orders of magnitude) than the human enzyme . In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function . Specific 12ß-Hydroxylation of Cinobufagin by Filamentous Fungi. Min Ye, 2004.Biotransformation of natural products has great potential for producing new drugs and could provide in vitro models of mammalian metabolism . Microbial transformation of the cytotoxic steroid cinobufagin was investigated . Cinobufagin could be specifically hydroxylated at the 12ß-position by the fungus Alternaria alternata . Six products from a scaled-up fermentation were obtained by silica gel column chromatography and reversed-phase liquid chromatography and were identified as 12ß-hydroxyl cinobufagin, 12ß-hydroxyl desacetylcinobufagin, 3-oxo-12ß-hydroxyl cinobufagin, 3-oxo-12ß-hydroxyl desacetylcinobufagin, 12-oxo-cinobufagin, and 3-oxo-12  -hydroxyl cinobufagin . The last five products are new compounds . 12ß-Hydroxylation of cinobufagin by A . alternata is a fast catalytic reaction and was complete within 8 h of growth with the substrate . This reaction was followed by dehydrogenation of the 3-hydroxyl group and then deacetylation at C-16 . Hydroxylation at C-12ß also was the first step in the metabolism of cinobufagin by a variety of fungal strains . In vitro cytotoxicity assays suggest that 12ß-hydroxyl cinobufagin and 3-oxo-12-hydroxyl cinobufagin exhibit somewhat decreased but still significant cytotoxic activities . The 12ß-hydroxylated bufadienolides produced by microbial transformation are difficult to obtain by chemical synthesis .
Nitric Oxide Metabolism in Neisseria meningitidis. Muna F. Anjum, 2002.Neisseria meningitidis, the causative agent of meningococcal disease in humans, is likely to be exposed to nitrosative stress during natural colonization and disease . The genome of N . meningitidis includes the genes aniA and norB, predicted to encode nitrite reductase and nitric oxide (NO) reductase, respectively . These gene products should allow the bacterium to denitrify nitrite to nitrous oxide . We show that N . meningitidis can support growth microaerobically by the denitrification of nitrite via NO and that norB is required for anaerobic growth with nitrite . NorB and, to a lesser extent, the cycP gene product cytochrome c' are able to counteract toxicity due to exogenously added NO . Expression of these genes by N . meningitidis during colonization and disease may confer protection against exogenous or endogenous nitrosative stress . Functional Analysis of HrpF, a Putative Type III Translocon Protein from Xanthomonas campestris pv . vesicatoria. Daniela Büttner, 2002.Type III secretion systems (TTSSs) are specialized protein transport systems in gram-negative bacteria which target effector proteins into the host cell . The TTSS of the plant pathogen Xanthomonas campestris pv . vesicatoria, encoded by the hrp (hypersensitive reaction and pathogenicity) gene cluster, is essential for the interaction with the plant . One of the secreted proteins is HrpF, which is required for pathogenicity but dispensable for type III secretion of effector proteins in vitro, suggesting a role in translocation . In this study, complementation analyses of an hrpF null mutant strain using various deletion derivatives revealed the functional importance of the C-terminal hydrophobic protein region . Deletion of the N terminus abolished type III secretion of HrpF . Employing the type III effector AvrBs3 as a reporter, we show that the N terminus of HrpF contains a signal for secretion but not a functional translocation signal . Experiments with lipid bilayers revealed a lipid-binding activity of HrpF as well as HrpF-dependent pore formation . These data indicate that HrpF presumably plays a role at the bacterial-plant interface as part of a bacterial translocon which mediates effector protein delivery across the host cell membrane .
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