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Presence of a Novel Phosphopentomutase and a 2-Deoxyribose 5-Phosphate Aldolase Reveals a Metabolic Link between Pentoses and Central Carbon Metabolism in the Hyperthermophilic Archaeon Thermococcus kodakaraensis. Naeem Rashid, 2004.Numerous bacteria and mammalian cells harbor two enzymes, phosphopentomutase (PPM) and 2-deoxyribose 5-phosphate aldolase (DERA), involved in the interconversion between nucleosides and central carbon metabolism . In this study, we have examined the presence of this metabolic link in the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 . A search of the genome sequence of this strain revealed the presence of a closely related orthologue (TK2104) of bacterial DERA genes while no orthologue related to previously characterized PPM genes could be detected . Expression, purification, and characterization of the TK2104 protein product revealed that this gene actually encoded a DERA, catalyzing the reaction through a class I aldolase mechanism . As PPM activity was detected in T . kodakaraensis cells, we partially purified the protein to examine its N-terminal amino acid sequence . The sequence corresponded to a gene (TK1777) similar to phosphomannomutases within COG1109 but not COG1015, which includes all previously identified PPMs . Heterologous gene expression of TK1777 and characterization of the purified recombinant protein clearly revealed that the gene indeed encoded a PPM . Both enzyme activities could be observed in T . kodakaraensis cells under glycolytic and gluconeogenic growth conditions, whereas the addition of ribose, 2-deoxyribose, and 2'-deoxynucleosides in the medium did not lead to a significant induction of these activities . Our results clearly indicate the presence of a metabolic link between pentoses and central carbon metabolism in T . kodakaraensis, providing an alternative route for pentose biosynthesis through the functions of DERA and a structurally novel PPM . Use of Fluorescent Lectin Probes for Analysis of Footprints from Pseudomonas aeruginosa MDC on Hydrophilic and Hydrophobic Glass Substrata. Eduardo Mora Bejarano, 2004. The Crystal Structure of Shikimate Dehydrogenase (AroE) Reveals a Unique NADPH Binding Mode. Sheng Ye, 2003.Shikimate dehydrogenase catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate . We report the first X-ray structure of shikimate dehydrogenase from Haemophilus influenzae to 2.4-Å resolution and its complex with NADPH to 1.95-Å resolution . The molecule contains two domains, a catalytic domain with a novel open twisted  /ß motif and an NADPH binding domain with a typical Rossmann fold . The enzyme contains a unique glycine-rich P-loop with a conserved sequence motif, GAGGXX, that results in NADPH adopting a nonstandard binding mode with the nicotinamide and ribose moieties disordered in the binary complex . A deep pocket with a narrow entrance between the two domains, containing strictly conserved residues primarily contributed by the catalytic domain, is identified as a potential 3-dehydroshikimate binding pocket . The flexibility of the nicotinamide mononucleotide portion of NADPH may be necessary for the substrate 3-dehydroshikimate to enter the pocket and for the release of the product shikimate .
Functional Domains of the RhlR Transcriptional Regulator of Pseudomonas aeruginosa. Janet R. Lamb, 2003.The RhlR transcriptional regulator of Pseudomonas aeruginosa, along with its cognate autoinducer, N-butyryl homoserine lactone (C4-HSL), regulates gene expression in response to cell density . With an Escherichia coli LexA-based protein interaction system, we demonstrated that RhlR multimerized and that the degree of multimerization was dependent on the C4-HSL concentration. Studies with an E . coli lasB::lacZ lysogen demonstrated that RhlR multimerization was necessary for it to function as a transcriptional activator . Deletion analysis of RhlR indicated that the N-terminal domain of the protein is necessary for C4-HSL binding . Single amino acid substitutions in the C-terminal domain of RhlR generated mutant RhlR proteins that had the ability to bind C4-HSL and multimerize but were unable to activate lasB expression, demonstrating that the C-terminal domain is important for target gene activation . Single amino acid substitutions in both the N-terminal and C-terminal domains of RhlR demonstrated that both domains possess residues involved in multimerization . RhlR with a C-terminal deletion and an RhlR site-specific mutant form that possessed multimerization but not transcriptional activation capabilities were able to inhibit the ability of wild-type RhlR to activate rhlA expression in P . aeruginosa. We conclude that C4-HSL binding is necessary for RhlR multimerization and that RhlR functions as a multimer in P. aeruginosa . Dual Resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa Toxins in Heliothis virescens Suggests Multiple Mechanisms of Resistance. Juan Luis Jurat-Fuentes, 2003.One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking) . This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests . In B . thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm) . After previous laboratory selection with Cry1Ac, H . virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa . We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains . In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects . Since Cry1Aa and Cry2Aa do not share binding proteins in H . virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding . Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected . These results highlight the genetic potential of H . virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance .
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