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Saturation Mutagenesis of Burkholderia cepacia R34 2,4-Dinitrotoluene Dioxygenase at DntAc Valine 350 for Synthesizing Nitrohydroquinone, Methylhydroquinone, and Methoxyhydroquinone. Brendan G. Keenan, 2004.Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc  -subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates) . Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol . Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography . V350F produced both nitrohydroquinone at a rate of 0.75 ± 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 ± 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 ± 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 ± 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 ± 0.02 nmol/min/mg of protein, both methylhydroquinone at 1.52 ± 0.02 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.74 ± 0.05 nmol/min/mg of protein from o-cresol, and methylhydroquinone at 0.43 ± 0.1 nmol/min/mg of protein from m-cresol . V350M produced both nitrohydroquinone at a rate of 0.33 nmol/min/mg of protein and 3-nitrocatechol at 0.089 nmol/min/mg of protein from o-nitrophenol, methoxyhydroquinone from o-methoxyphenol at 2.4 nmol/min/mg of protein, methylhydroquinone at 1.97 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.11 nmol/min/mg of protein from o-cresol . The DDO variants V350F and V350M also exhibited 10-fold-enhanced activity towards naphthalene (8 ± 2.6 nmol/min/mg of protein), forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene . Hence, mutagenesis of wild-type DDO through active-site engineering generated variants with relatively high rates toward a previously uncharacterized class of substituted phenols for the nitroarene dioxygenases; seven previously uncharacterized substrates were evaluated for wild-type DDO, and four novel monooxygenase-like products were found for the DDO variants V350F and V350M (methoxyhydroquinone, methylhydroquinone, 2-hydroxybenzyl alcohol, and 3-nitrocatechol) .
Properties of the Glucose-6-Phosphate Transporter from Chlamydia pneumoniae (HPTcp) and the Glucose-6-Phosphate Sensor from Escherichia coli (UhpC). Christian Schwöppe, 2002.The amino acid sequence of the proposed glucose-6-phosphate (Glc6P) transporter from Chlamydia pneumoniae (HPTcp; hexose phosphate transporter [Chlamydia pneumoniae]) exhibits a higher degree of similarity to the Escherichia coli Glc6P sensor (UhpC) than to the E . coli Glc6P transporter (UhpT) . Overexpression of His-UhpC in a UhpT-deficient E . coli strain revealed that the sensor protein is also able to transport Glc6P and exhibits an apparent Km (Glc6P) of 25 µM, whereas His-HPTcp exhibits an apparent Km (Glc6P) of 98 µM . His-HPTcp showed a four-times-lower specific activity than His-UhpT but a 56-times-higher specific activity than His-UhpC . Like His-UhpT and His-UhpC, the carrier His-HPTcp performs a sugar-phosphate/inorganic-phosphate antiporter mode of transport . Surprisingly, while physiological concentrations of inorganic phosphate competitively inhibited transport mediated by the E . coli proteins His-UhpT and His-UhpC, transport mediated by His-HPTcp was not inhibited . Interestingly, C3-organophosphates stimulated His-HPTcp activity but not His-UhpT- or His-UhpC-catalyzed Glc6P transport . In contrast to His-UhpC, the His-HPTcp protein does not act as a Glc6P sensor in the uhp regulon . Substrate Specificity Classes and the Recognition Signal for Salmonella Type III Flagellar Export. Takanori Hirano, 2003.Most flagellar proteins of Salmonella are exported to their assembly destination via a specialized apparatus . This apparatus is a member of the type III superfamily, which is widely used for secretion of virulence factors by pathogenic bacteria . Extensive studies have been carried out on the export of several of the flagellar proteins, most notably the hook protein (FlgE), the hook-capping protein (FlgD), and the filament protein flagellin (FliC) . This has led to the concept of two export specificity classes, the rod/hook type and the filament type . However, little direct experimental evidence has been available on the export properties of the basal-body rod proteins (FlgB, FlgC, FlgF, and FlgG), the putative MS ring-rod junction protein (FliE), or the muramidase and putative rod-capping protein (FlgJ) . In this study, we have measured the amounts of these proteins exported before and after hook completion . Their amounts in the culture supernatant from a flgE mutant (which is still at the hook-type specificity stage) were much higher than those from a flgK mutant (which has advanced to the filament-type specificity stage), placing them in the same class as the hook-type proteins . Overproduction of FliE, FlgB, FlgC, FlgF, FlgG, or FlgJ caused inhibition of the motility of wild-type cells and inhibition of the export of the hook-capping protein FlgD . We also examined the question of whether export and translation are linked and found that all substrates tested could be exported after protein synthesis had been blocked by spectinomycin or chloramphenicol . We conclude that the amino acid sequence of these proteins suffices to mediate their recognition and export .
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