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The Phosphoenolpyruvate Carboxylase from Methanothermobacter thermautotrophicus Has a Novel Structure. Hiten M. Patel, 2004.In Methanothermobacter thermautotrophicus, oxaloacetate synthesis is a major and essential CO2-fixation reaction . This methanogenic archaeon possesses two oxaloacetate-synthesizing enzymes, pyruvate carboxylase and phosphoenolpyruvate carboxylase . The phosphoenolpyruvate carboxylase from this organism was purified to homogeneity . The subunit size of this homotetrameric protein was 55 kDa, which is about half that of all known bacterial and eukaryotic phosphoenolpyruvate carboxylases (PPCs) . The NH2-terminal sequence identified this enzyme as the product of MTH943, an open reading frame with no assigned function in the genome sequence . A BLAST search did not show an obvious sequence similarity between MTH943 and known PPCs, which are generally well conserved . This is the first report of a new type of phosphoenolpyruvate carboxylase that we call PpcA ("A" for "archaeal") . Homologs to PpcA were present in most archaeal genomic sequences, but only in three bacterial (Clostridium perfringens, Oenococcus oeni, and Leuconostoc mesenteroides) and no eukaryotic genomes . PpcA was the only recognizable oxaloacetate-producing enzyme in Methanopyrus kandleri, a hydrothermal vent organism . Each PpcA-containing organism lacked a PPC homolog . The activity of M . thermautotrophicus PpcA was not influenced by acetyl coenzyme A and was about 50 times less sensitive to aspartate than the Escherichia coli PPC . The catalytic core (including His138, Arg587, and Gly883) of the E . coli PPC was partly conserved in PpcA, but three of four aspartate-binding residues (Lys773, Arg832, and Asn881) were not . PPCs probably evolved from PpcA through a process that added allosteric sites to the enzyme . The reverse is also equally possible . Escherichia coli DNA Polymerase III Can Replicate Efficiently past a T-T cis-syn Cyclobutane Dimer if DNA Polymerase V and the 3' to 5' Exonuclease Proofreading Function Encoded by dnaQ Are Inactivated. Angela Borden, 2002.Although very little replication past a T-T cis-syn cyclobutane dimer normally takes place in Escherichia coli in the absence of DNA polymerase V (Pol V), we previously observed as much as half of the wild-type bypass frequency in Pol V-deficient (  umuDC) strains if the 3' to 5' exonuclease proofreading activity of the Pol III
 subunit was also disabled by mutD5 . This observation might be explained in at least two ways . In the absence of Pol V, wild-type Pol III might bind preferentially to the blocked primer terminus but be incapable of bypass, whereas the proofreading-deficient enzyme might dissociate more readily, providing access to bypass polymerases . Alternatively, even though wild-type Pol III is generally regarded as being incapable of lesion bypass, proofreading-impaired Pol III might itself perform this function . We have investigated this issue by examining dimer bypass frequencies in
umuDC mutD5 strains that were also deficient for Pol I, Pol II, and Pol IV, both singly and in all combinations . Dimer bypass frequencies were not decreased in any of these strains and indeed in some were increased to levels approaching those found in strains containing Pol V . Efficient dimer bypass was, however, entirely dependent on the proofreading deficiency imparted by mutD5, indicating the surprising conclusion that bypass was probably performed by the mutD5 Pol III enzyme itself . This mutant polymerase does not replicate past the much more distorted T-T (6-4) photoadduct, however, suggesting that it may only replicate past lesions, like the T-T dimer, that form base pairs normally .
VmrA, a Member of a Novel Class of Na+-Coupled Multidrug Efflux Pumps from Vibrio parahaemolyticus. Jing Chen, 2002.Gene vmrA, cloned from Vibrio parahaemolyticus, made Escherichia coli resistant to 4prime;,6-diamino-2-phenylindol, tetraphenylphosphonium chloride, acriflavine, and ethidium bromide . VmrA belongs to the DinF branch of MATE family efflux transporters . VmrA catalyzed acriflavine efflux and showed Na+/drug transporter activity because the addition of tetraphenylphosphonium to Na+-loaded cells caused Na+ efflux . Overproduction of Thermus sp . Strain T2 ß-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports. Benevides C. C. Pessela, 2003.A novel thermostable chimeric ß-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the ß-galactosidase from Thermus sp . strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC) . The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the ß-galactosidase . Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability . To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports . The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports . Yeast Genome-Wide Expression Analysis Identifies a Strong Ergosterol and Oxidative Stress Response during the Initial Stages of an Industrial Lager Fermentation. Vincent J. Higgins, 2003.Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae during the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection . The induction of the ERG genes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period . From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage . Additionally, similar ERG10 and TRR1 gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response . Further analysis showed that erg mutants producing altered sterols were highly sensitive to oxidative stress-generating compounds . Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation .
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