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Probing Conservation of HAMP Linker Structure and Signal Transduction Mechanism through Analysis of Hybrid Sensor Kinases. J. Alex Appleman, 2003.The HAMP linker, a predicted structural element observed in many sensor kinases and methyl-accepting chemotaxis proteins, transmits signals between sensory input modules and output modules . HAMP linkers are located immediately inside the cytoplasmic membrane and are predicted to form two short amphipathic  -helices (AS-1 and AS-2) joined by an unstructured connector . HAMP linkers are found in the Escherichia coli nitrate- and nitrite-responsive sensor kinases NarX and NarQ (which respond to ligand by increasing kinase activity) and the sensor kinase CpxA (which responds to ligand by decreasing kinase activity) . We constructed a series of hybrids with fusion points throughout the HAMP linker, in which the sensory modules of NarX or NarQ are fused to the transmitter modules of NarX, NarQ, or CpxA . A hybrid of the NarX sensor module and the CpxA HAMP linker and transmitter module (NarX-CpxA-1) responded to nitrate by decreasing kinase activity, whereas a hybrid in which the HAMP linker of NarX was replaced by that of CpxA (NarX-CpxA-NarX-1) responded to nitrate by increasing kinase activity . However, sequence variations between HAMP linkers do not allow free exchange of HAMP linkers or their components . Certain deletions in the NarX HAMP linker resulted in characteristic abnormal responses to ligand; similar deletions in the NarQ and NarX-CpxA-1 HAMP linkers resulted in responses to ligand generally similar to those seen in NarX . We conclude that the structure and action of the HAMP linker are conserved and that the HAMP linker transmits a signal to the output domain that ligand is bound .
QscR, a LysR-Type Transcriptional Regulator and CbbR Homolog, Is Involved in Regulation of the Serine Cycle Genes in Methylobacterium extorquens AM1. Marina G. Kalyuzhnaya, 2003.A new gene, qscR, encoding a LysR-type transcriptional regulator that is a homolog of CbbR, has been characterized from the facultative methylotroph Methylobacterium extorquens AM1 and shown to be the major regulator of the serine cycle, the specific C1 assimilation pathway . The qscR mutant was shown to be unable to grow on C1 compounds, and it lacked the activity of serine-glyoxylate aminotransferase, a key enzyme of the serine cycle . Activities of other serine cycle enzymes were decreased during growth on C1 compounds compared to the activities found in wild-type M . extorquens AM1 . Promoter fusion assays, as well as reverse transcription-PCR assays, have indicated that the serine cycle genes belong to three separate transcriptional units, sga-hpr-mtdA-fch, mtkA-mtkB-ppc-mcl, and gly . Gel retardation assays involving the purified QscR have demonstrated the specific binding of QscR to the DNA regions upstream of sga, mtkA, gly, and qscR . We conclude that QscR acts as a positive transcriptional regulator of most of the serine cycle enzymes and also as an autorepressor . Small, Acid-Soluble Proteins as Biomarkers in Mass Spectrometry Analysis of Bacillus Spores. Yetrib Hathout, 2003.The use of 1 N HCl for extraction of small, acid-soluble proteins (SASP) from different Bacillus spore species was examined . The extracts were analyzed by high-performance liquid chromatography and matrix-assisted laser desorption mass spectrometry and were found to be both qualitatively and quantitatively superior to extraction by acetonitrile-5% trifluoroacetic acid (70:30, vol/vol) . Both major and minor  /ß- and
 -type SASP were characterized by their molecular masses or tryptic peptide maps and by searches of both protein and unannotated genome databases . For all but 1 pair (B . cereus T and B . thuringiensis subsp . Kurstaki) among the 11 variants studied the suites of SASP masses are distinctive, consistent with the use of these proteins as potential biomarkers for spore identification by mass spectrometry .
Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae. Jung-Kul Lee, 2003.Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol . Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics . In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis . The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric . Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases . C . magnoliae erythrose reductase catalyzes the reduction of various aldehydes . Among aldoses, erythrose was the preferred substrate (Km = 7.9 mM; kcat/Km = 0.73 mM-1 s-1) . This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (kcat/Km = 450 mM-1 s-1) than with NADPH (kcat/Km = 5.5 mM-1 s-1), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily . Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism . The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu2+ and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase . These data indicate that the C . magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity .
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