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101.
102.
AhpF of Salmonella typhimurium, the flavoprotein reductase required for catalytic turnover of AhpC with hydroperoxide substrates in the alkyl hydroperoxide reductase system, is a 57 kDa protein with homology to thioredoxin reductase (TrR) from Escherichia coli. Like TrR, AhpF employs tightly bound FAD and redox-active disulfide center(s) in catalyzing electron transfer from reduced pyridine nucleotides to the disulfide bond of its protein substrate. Homology of AhpF to the smaller (35 kDa) TrR protein occurs in the C-terminal part of AhpF; a stretch of about 200 amino acids at the N-terminus of AhpF contains an additional redox-active disulfide center and is required for catalysis of AhpC reduction. We have demonstrated that fusion of the N-terminal 207 amino acids of AhpF to full-length TrR results in a chimeric protein (Nt-TrR) with essentially the same catalytic efficiency (k(cat)/K(m)) as AhpF in AhpC reductase assays; both k(cat) and the K(m) for AhpC are decreased about 3-4-fold for Nt-TrR compared with AhpF. In addition, Nt-TrR retains essentially full TrR activity. Based on results from two mutants of Nt-TrR (C129, 132S and C342,345S), AhpC reductase activity requires both centers while TrR activity requires only the C-terminal-most disulfide center in Nt-TrR. The high catalytic efficiency with which Nt-TrR can reduce thioredoxin implies that the attached N-terminal domain does not block access of thioredoxin to the TrR-derived Cys342-Cys345 center of Nt-TrR nor does it impede the putative conformational changes that this part of Nt-TrR is proposed to undergo during catalysis. These studies indicate that the C-terminal part of AhpF and bacterial TrR have very similar mechanistic properties. These findings also confirm that the N-terminal domain of AhpF plays a direct role in AhpC reduction. 相似文献
103.
Bailey JK Kindig CA Behnke BJ Musch TI Schmid-Schoenbein GW Poole DC 《American journal of physiology. Heart and circulatory physiology》2000,279(6):H3131-H3137
Intravital microscopy facilitates insights into muscle microcirculatory structural and functional control, provided that surgical exteriorization does not impact vascular function. We utilized a novel combination of phosphorescence quenching, microvascular oxygen pressure (microvascular PO(2)), and microsphere (blood flow) techniques to evaluate static and dynamic behavior within the exposed intact (I) and exteriorized (EX) rat spinotrapezius muscle. I and EX muscles were studied under control, metabolic blockade with 2,4-dinitrophenol (DNP), and electrically stimulated conditions with 1-Hz contractions, and across switches from 21 to 100% and 10% inspired O(2). Surgical preparation did not alter spinotrapezius muscle blood flow in either I or EX muscle. DNP elevated muscle blood flow approximately 120% (P < 0.05) in both I and EX muscles (P > 0.05 between I and EX). Contractions reduced microvascular PO(2) from 30.4 +/- 4.3 to 21.8 +/- 4.8 mmHg in I muscle and from 33.2 +/- 3.0 to 25.9 +/- 2.8 mmHg in EX muscles with no difference between I and EX. In each O(2) condition, there was no difference (each P > 0.05) in microvascular PO(2) between I and EX muscles (21% O(2): I = 37 +/- 1; EX = 36 +/- 1; 100%: I = 62 +/- 5; EX = 51 +/- 9; 10%: I = 20 +/- 1; EX = 17 +/- 2 mmHg). Similarly, the dynamic behavior of microvascular PO(2) to altered inspired O(2) was unaffected by the EX procedure [half-time (t(1/2)) to 100% O(2): I = 23 +/- 5; EX = 23 +/- 4; t(1/2) to 10%: I = 14 +/- 2; EX = 16 +/- 2 s, both P > 0.05]. These results demonstrate that the spinotrapezius muscle can be EX without significant alteration of microvascular integrity and responsiveness under the conditions assessed. 相似文献
104.
105.
Fang QQ Mitchell A Regier JC Mitter C Friedlander TP Poole RW 《Molecular phylogenetics and evolution》2000,15(3):473-486
Phylogenetic utility for the nuclear gene encoding dopa decarboxylase (DDC), little used in systematics, was recently demonstrated within the noctuid moth subfamily Heliothinae. Here we extend the test of the utility of a 709-bp DDC fragment to deeper levels, analyzing 49 species representing major groups across the superfamily Noctuoidea. Parsimony, distance, and maximum-likelihood analyses recover all or nearly all of a set of "test clades" supported by clear morphological synapomorphies, spanning a wide range of taxonomic levels. DDC also upholds a recent proposal that the Noctuidae are paraphyletic. Nt3 contributes a majority of the signal and recovers the basal split between Notodontidae and all other noctuoids, despite a plateau of nt3 divergence at this level. However, nonsynonymous changes also support groups at all levels, and in contrast to nt3, amino acid divergence shows no plateau. The utility of DDC promises to extend back to the early Tertiary and Cretaceous, a time span for which few suitable genes have been identified. 相似文献
106.
107.
AhpF, the flavin-containing component of the Salmonella typhimurium alkyl hydroperoxide reductase system, catalyzes the NADH-dependent reduction of an active-site disulfide bond in the other component, AhpC, which in turn reduces hydroperoxide substrates. The amino acid sequence of the C-terminus of AhpF is 35% identical to that of thioredoxin reductase (TrR) from Escherichia coli. AhpF contains an additional 200-residue N-terminal domain possessing a second redox-active disulfide center also required for AhpC reduction. Our studies indicate that this N-terminus contains a tandem repeat of two thioredoxin (Tr)-like folds, the second of which contains the disulfide redox center. Structural and catalytic properties of independently expressed fragments of AhpF corresponding to the TrR-like C-terminus (F[208-521]) and the 2Tr-like N-terminal domain (F[1-202]) have been addressed. Enzymatic assays, reductive titrations, and circular dichroism studies of the fragments indicate that each folds properly and retains many functional properties. Electron transfer between F[208-521] and F[1-202] is, however, relatively slow (4 x 10(4) M(-)(1) s(-)(1) at 25 degrees C) and nonsaturable up to 100 microM F[1-202]. TrR is nearly as efficient at F[1-202] reduction as is F[208-521], although neither the latter fragment, nor intact AhpF, can reduce Tr. An engineered mutant AhpC substrate with a fluorophore attached via a disulfide bond has been used to demonstrate that only F[1-202], and not F[208-521], is capable of electron transfer to AhpC, thereby establishing the direct role this N-terminal domain plays in mediating electron transfer between the TrR-like part of AhpF and AhpC. 相似文献
108.
Influence of the MexAB-OprM Multidrug Efflux System on Quorum Sensing in Pseudomonas aeruginosa 总被引:6,自引:0,他引:6 下载免费PDF全文
Kelly Evans Luciano Passador Ramakrishnan Srikumar Eric Tsang Jonathon Nezezon Keith Poole 《Journal of bacteriology》1998,180(20):5443-5447
Pseudomonas aeruginosa nalB mutants which hyperexpress the MexAB-OprM multidrug efflux system produce reduced levels of several extracellular virulence factors known to be regulated by quorum sensing. Such mutants also produce less acylated homoserine lactone autoinducer PAI-1, consistent with an observed reduction in lasI expression. These data suggest that PAI-1 is a substrate for MexAB-OprM, and its resulting exclusion from cells hyperexpressing MexAB-OprM limits PAI-1-dependent activation of lasI and the virulence genes. 相似文献
109.
Identification and Characterization of aarF, a Locus Required for Production of Ubiquinone in Providencia stuartii and Escherichia coli and for Expression of 2′-N-Acetyltransferase in P. stuartii 下载免费PDF全文
David R. Macinga Gregory M. Cook Robert K. Poole Philip N. Rather 《Journal of bacteriology》1998,180(1):128-135
Providencia stuartii contains a chromosomal 2′-N-acetyltransferase [AAC(2′)-Ia] involved in the O acetylation of peptidoglycan. The AAC(2′)-Ia enzyme is also capable of acetylating and inactivating certain aminoglycosides and confers high-level resistance to these antibiotics when overexpressed. We report the identification of a locus in P. stuartii, designated aarF, that is required for the expression of AAC(2′)-Ia. Northern (RNA) analysis demonstrated that aac(2′)-Ia mRNA levels were dramatically decreased in a P. stuartii strain carrying an aarF::Cm disruption. The aarF::Cm disruption also resulted in a deficiency in the respiratory cofactor ubiquinone. The aarF locus encoded a protein that had a predicted molecular mass of 62,559 Da and that exhibited extensive amino acid similarity to the products of two adjacent open reading frames of unknown function (YigQ and YigR), located at 86 min on the Escherichia coli chromosome. An E. coli yigR::Kan mutant was also deficient in ubiquinone content. Complementation studies demonstrated that the aarF and the E. coli yigQR loci were functionally equivalent. The aarF or yigQR genes were unable to complement ubiD and ubiE mutations that are also present at 86 min on the E. coli chromosome. This result indicates that aarF (yigQR) represents a novel locus for ubiquinone production and reveals a previously unreported connection between ubiquinone biosynthesis and the regulation of gene expression. 相似文献
110.
Role of the Multidrug Efflux Systems of Pseudomonas aeruginosa in Organic Solvent Tolerance 总被引:6,自引:0,他引:6 下载免费PDF全文
Multidrug efflux pumps with a broad substrate specificity make a major contribution to intrinsic and acquired multiple antibiotic resistance in Pseudomonas aeruginosa. Using genetically defined efflux pump mutants, we investigated the involvement of the three known efflux systems, MexA-MexB-OprM, MexC-MexD-OprJ, and MexE-MexF-OprN, in organic solvent tolerance in this organism. Our results showed that all three systems are capable of providing some level of tolerance to organic solvents such as n-hexane and p-xylene. Expression of MexAB-OprM was correlated with the highest levels of tolerance, and indeed, this efflux system was a major contributor to the intrinsic solvent tolerance of P. aeruginosa. Intrinsic organic solvent tolerance was compromised by a protonophore, indicating that it is substantially energy dependent. These data suggest that the efflux of organic solvents is a factor in the tolerance of P. aeruginosa to these compounds and that the multidrug efflux systems of this organism can accommodate organic solvents, as well as antibiotics. 相似文献