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Characterization of Interactions between the Transcriptional Repressor PhlF and Its Binding Site at the phlA Promoter in Pseudomonas fluorescens F113. Abdelhamid Abbas, 2002.The phlACBD genes responsible for the biosynthesis of the antifungal metabolite 2,4-diacetylphloroglucinol (PHL) by the biocontrol strain Pseudomonas fluorescens F113 are regulated at the transcriptional level by the pathway-specific repressor PhlF . Strong evidence suggests that this regulation occurs mainly in the early logarithmic phase of growth . First, the expression of the phlF gene is relatively high between 3 and 13 h of growth and relatively low thereafter, with the phlACBD operon following an opposite expression profile . Second, the kinetics of PHL biosynthesis are specifically altered in the logarithmic phase in a P . fluorescens F113 phlF mutant . The phlA-phlF intergenic region presents a complex organization in that phlACBD is transcribed from a  70 RNA polymerase-dependent promoter that is likely to overlap the promoter of the divergently transcribed phlF gene . The repression by PhlF is due to its interaction with an inverted repeated sequence, phO, located downstream of the phlA transcriptional start site . Cross-linking experiments indicate that PhlF can dimerize in solution, and thus PhlF may bind phO as a dimer or higher-order complex . Furthermore, it is now demonstrated that certain regulators of PHL synthesis act by modulating PhlF binding to phO . PHL, which has previously been shown to be an autoinducer of PHL biosynthesis, interacts with PhlF to destabilize the PhlF-phO complex . Conversely, the PhlF-phO complex is stabilized by the presence of salicylate, which has been shown to be an inhibitor of phlA expression .
Malonyl-Coenzyme A Reductase from Chloroflexus aurantiacus, a Key Enzyme of the 3-Hydroxypropionate Cycle for Autotrophic CO2 Fixation. Michael Hügler, 2002.The 3-hydroxypropionate cycle is a new autotrophic CO2 fixation pathway in Chloroflexus aurantiacus and some archaebacteria . The initial step is acetyl-coenzyme A (CoA) carboxylation to malonyl-CoA by acetyl-CoA carboxylase, followed by NADPH-dependent reduction of malonyl-CoA to 3-hydroxypropionate . This reduction step was studied in Chloroflexus aurantiacus . A new enzyme was purified, malonyl-CoA reductase, which catalyzed the two-step reduction malonyl-CoA + NADPH + H+  malonate semialdehyde + NADP+ + CoA and malonate semialdehyde + NADPH + H+
3-hydroxypropionate + NADP+ . The bifunctional enzyme (aldehyde dehydrogenase and alcohol dehydrogenase) had a native molecular mass of 300 kDa and consisted of a single large subunit of 145 kDa, suggesting an
 2 composition . The N-terminal amino acid sequence was determined, and the incomplete gene was identified in the genome database . Obviously, the enzyme consists of an N-terminal short-chain alcohol dehydrogenase domain and a C-terminal aldehyde dehydrogenase domain . No indication of the presence of a prosthetic group was obtained; Mg2+ and Fe2+ stimulated and EDTA inhibited activity . The enzyme was highly specific for its substrates, with apparent Km values of 30 µM malonyl-CoA and 25 µM NADPH and a turnover number of 25 s-1 subunit-1 . The specific activity in autotrophically grown cells was 0.08 µmol of malonyl-CoA reduced min-1 (mg of protein)-1, compared to 0.03 µmol min-1 (mg of protein)-1 in heterotrophically grown cells, indicating downregulation under heterotrophic conditions . Malonyl-CoA reductase is not required in any other known pathway and therefore can be taken as a characteristic enzyme of the 3-hydroxypropionate cycle . Furthermore, the enzyme may be useful for production of 3-hydroxypropionate and for a coupled spectrophotometric assay for activity screening of acetyl-CoA carboxylase, a target enzyme of potent herbicides .
Changes in the Concentrations of Guanosine 5'-Diphosphate 3'-Diphosphate and the Initiating Nucleoside Triphosphate Account for Inhibition of rRNA Transcription in Fructose-1,6-Diphosphate Aldolase (fda) Mutants. David A. Schneider, 2003.Early screens for conditional lethal mutations that affected rRNA expression in Escherichia coli identified temperature-sensitive fda mutants (fda encodes the glycolytic enzyme fructose-1,6-diphosphate aldolase) . It was shown that these fda(Ts) mutants were severely impaired in rRNA synthesis upon shift to the restrictive temperature, although the mechanism of inhibition was never determined . Here, we bring resolution to this long-standing question by showing that changes in the concentrations of guanosine 5'-diphosphate 3'-diphosphate and initiating nucleoside triphosphates can account for the previously observed effects of fda mutations on rRNA transcription .
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