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Hazard Ratio in Clinical Trials. Spotswood L. Spruance, 2004. The First Archaeal ATP-Dependent Glucokinase, from the Hyperthermophilic Crenarchaeon Aeropyrum pernix, Represents a Monomeric, Extremely Thermophilic ROK Glucokinase with Broad Hexose Specificity. Thomas Hansen, 2002.An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix was purified 230-fold to homogeneity . The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa . The apparent Km values for ATP and glucose (at 90°C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent Vmax was about 35 U/mg . The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (kcat/Km), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose . Divalent cations were required for maximal activity: Mg2+, which was most effective, could partially be replaced with Co2+, Mn2+, and Ni2+ . The enzyme had a temperature optimum of at least 100°C and showed significant thermostability up to 100°C . The coding function of open reading frame (ORF) APE2091 (Y . Kawarabayasi, Y . Hino, H . Horikawa, S . Yamazaki, Y . Haikawa, K . Jin-no, M . Takahashi, M . Sekine, S . Baba, A . Ankai, H . Kosugi, A . Hosoyama, S . Fukui, Y . Nagai, K . Nishijima, H . Nakazawa, M . Takamiya, S . Masuda, T . Funahashi, T . Tanaka, Y . Kudoh, J . Yamazaki, N . Kushida, A . Oguchi, and H . Kikuchi, DNA Res . 6:83-101, 145-152, 1999), previously annotated as gene glk, coding for ATP-glucokinase of A . pernix, was proved by functional expression in Escherichia coli . The purified recombinant ATP-dependent glucokinase showed a 5-kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from A . pernix. N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091 . The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which comprise bacterial sugar kinases and transcriptional repressors . This is the first report of the characterization of an ATP-dependent glucokinase from the domain of Archaea, which differs from its bacterial counterparts by its monomeric structure and its broad specificity for hexoses . Investigation of the Role of Electrostatic Charge in Activation of the Escherichia coli Response Regulator CheY. Jenny G. Smith, 2003.In a two-component regulatory system, an important means of signal transduction in microorganisms, a sensor kinase phosphorylates a response regulator protein on an aspartyl residue, resulting in activation . The active site of the response regulator is highly charged (containing a lysine, the phosphorylatable aspartate, two additional aspartates involved in metal binding, and an Mg2+ ion), and introduction of the dianionic phosphoryl group results in the repositioning of charged moieties . Furthermore, substitution of one of the Mg2+-coordinating aspartates with lysine or arginine in the Escherichia coli chemotaxis response regulator CheY results in phosphorylation-independent activation . In order to examine the consequences of altered charge distribution for response regulator activity and to identify possible additional amino acid substitutions that result in phosphorylation-independent activation, we made 61 CheY mutants in which residues close to the site of phosphorylation (Asp57) were replaced by various charged amino acids . Most substitutions (47 of 61) resulted in the complete loss of CheY activity, as measured by the inability to support clockwise flagellar rotation . However, 10 substitutions, all introducing a new positive charge, resulted in the loss of chemotaxis but in the retention of some clockwise flagellar rotation . Of the mutants in this set, only the previously identified CheY13DK and CheY13DR mutants displayed clockwise activity in the absence of the CheA sensor kinase . The absence of negatively charged substitution mutants with residual activity suggests that the introduction of additional negative charges into the active site is particularly deleterious for CheY function . Finally, the spatial distribution of positions at which amino acid substitutions are functionally tolerated or not tolerated is consistent with the presently accepted mechanism of response regulator activation and further suggests a possible role for Met17 in signal transduction by CheY . Positive Control of Swarming, Rhamnolipid Synthesis, and Lipase Production by the Posttranscriptional RsmA/RsmZ System in Pseudomonas aeruginosa PAO1. Karin Heurlier, 2004.In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules . RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P . aeruginosa . An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P . aeruginosa . A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P . aeruginosa . Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation . Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA .
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