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Double Mutant (double + mutant)
Terms modified by Double Mutant Selected AbstractsBacillus subtilis Esterase (BS2) and its Double Mutant Have Different Selectivity in the Removal of Carboxyl Protecting GroupsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 14-15 2009Efrosini Barbayianni Abstract An esterase from Bacillus subtilis (BS2) and its double mutant E188W/M193C quickly hydrolyze n -butyl, n -propyl, methoxyethyl and allyl esters. The wild-type BS2 preferentially removes such esters from the ,-position of glutamate diesters, while the engineered enzyme has a reversed selectivity removing esters from the ,-position of glutamate diesters. Automated docking and molecular dynamic simulations were performed to understand the molecular reason for the different regioselectivity. [source] Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomicsENVIRONMENTAL MICROBIOLOGY, Issue 12 2003Catalina Arevalo-Ferro Summary The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N -acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type. These results add haemoglobin utilization to the list of phenotypes controlled through QS in P. aeruginosa. The surprisingly high number of AHL-regulated proteins relative to the number of regulated genes suggests that quorum-sensing control also operates via post-transcriptional mechanisms. To strengthen this hypothesis we investigated the role of quorum sensing in the post-translational modification of HasAp, an extracellular protein required for the uptake of free and haemoglobin-bound haem. [source] Functional properties of the protein disulfide oxidoreductase from the archaeon Pyrococcus furiosusFEBS JOURNAL, Issue 16 2004A member of a novel protein family related to protein disulfide-isomerase Protein disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol,disulfide exchange reactions with a CXXC sequence motif at their active site. A disulfide oxidoreductase, a highly thermostable protein, was isolated from Pyrococcus furiosus (PfPDO), which is characterized by two redox sites (CXXC) and an unusual molecular mass. Its 3D structure at high resolution suggests that it may be related to the multidomain protein disulfide-isomerase (PDI), which is currently known only in eukaryotes. This work focuses on the functional characterization of PfPDO as well as its relation to the eukaryotic PDIs. Assays of oxidative, reductive, and isomerase activities of PfPDO were performed, which revealed that the archaeal protein not only has oxidative and reductive activity, but also isomerase activity. On the basis of structural data, two single mutants (C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites. The results indicate that the CPYC site in the C-terminal half of the protein is fundamental to reductive/oxidative activity, whereas isomerase activity requires both active sites. In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and an optimum temperature of 90 °C. These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel protein disulfide oxidoreductase family. [source] Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensisFEBS JOURNAL, Issue 3 2002Evert Bokma Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the ,1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower kcat and a slightly higher Km than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with ,,2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower kcat and an approximately twofold higher Km than the wild-type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild-type hevamine, the carbonyl oxygen atom of the N -acetyl group of the ,1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125,and Tyr183 contribute to catalysis by positioning the,carbonyl oxygen of the N -acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis. [source] Salmonella enteritidis temperature-sensitive mutants protect mice against challenge with virulent Salmonella strains of different serotypesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2000M.Magdalena Gherardi Abstract The protection conferred by temperature-sensitive mutants of Salmonella enteritidis against different wild-type Salmonella serotypes was investigated. Oral immunization with the single temperature-sensitive mutant E/1/3 or with a temperature-sensitive thymine-requiring double mutant (E/1/3T) conferred: (i) significant protection against the homologous wild-type Salmonella strains; (ii) significant cross-protection toward high challenge doses of S. typhimurium. Significant antibody levels against homologous lipopolysaccharide and against homologous and heterologous protein antigens were detected in sera from immunized mice. Moreover, a wide range of protein antigens from different Salmonella O serotypes were recognized by sera from immunized animals. Besides, primed lymphocytes from E/1/3 immunized mice recognized Salmonella antigens from different serotypes. Taken together, these results indicate that temperature-sensitive mutants of S. enteritidis are good candidates for the construction of live vaccines against Salmonella. [source] Stimulation of RNA polymerase II transcript cleavage activity contributes to maintain transcriptional fidelity in yeastGENES TO CELLS, Issue 5 2007Hiroshi Koyama The transcription elongation factor S-II, also designated TFIIS, stimulates the nascent transcript cleavage activity intrinsic to RNA polymerase II. Rpb9, a small subunit of RNA polymerase II, enhances the cleavage stimulation activity of S-II. Here, we investigated the role of nascent transcript cleavage stimulation activity on the maintenance of transcriptional fidelity in yeast. In yeast, S-II is encoded by the DST1 gene. Disruption of the DST1 gene decreased transcriptional fidelity in cells. Mutations in the DST1 gene that reduce the S-II cleavage stimulation activity led to decreased transcriptional fidelity in cells. A disruption mutant of the RPB9 gene also had decreased transcriptional fidelity. Expression of mutant Rpb9 proteins that are unable to enhance the S-II cleavage stimulation activity failed to restore the phenotype. These results suggest that both S-II and Rpb9 maintain transcriptional fidelity by stimulating the cleavage activity intrinsic to RNA polymerase II. Also, a DST1 and RPB9 double mutant had more severe transcriptional fidelity defect compared with the DST1 gene deletion mutant, suggesting that Rpb9 maintains transcriptional fidelity via two mechanisms, enhancement of S-II dependent cleavage stimulation and S-II independent function(s). [source] Expression of ribosome modulation factor (RMF) in Escherichia coli requires ppGppGENES TO CELLS, Issue 8 2001Kaori Izutsu Background During the transition from the logarithmic to the stationary phase, 70S ribosomes are dimerized into the 100S form, which has no translational activity. Ribosome Modulation Factor (RMF) is induced during the stationary phase and binds to the 50S ribosomal subunit, which directs the dimerization of 70S ribosomes. Unlike many other genes induced in the stationary phase, rmf transcription is independent of the sigma S. To identify the factors that regulate the growth phase-dependent induction of rmf, mutant strains deficient in global regulators were examined for lacZ expression directed by the rmf promoter. Results Among mutants of defective global regulators, only ppGpp deficiency (relA-spoT double mutant) drastically reduced the level of rmf transcription to less than 10% of that seen in the wild-type. Neither RMF nor 100S ribosomes were detected in this mutant in the stationary phase. rmf transcription correlated well with cellular ppGpp levels during amino acid starvation, IPTG induction of Ptrc-relA455 and in other mutants with artificially increased ppGpp levels. Although the growth rate also correlated inversely with both ppGpp levels and rmf transcription, the observation that the growth rates of the ppGpp-deficient and wild-type strains varied equivalently when grown on different media indicates that the link between rmf transcription and ppGpp levels is not a function of the growth rate. Conclusions ppGpp appears to positively regulate rmf transcription, at least in vivo. Thus, RMF provides a novel negative translational control by facilitating the formation of inactive ribosome dimers (100S) under the stringent circumstances of the stationary phase. [source] Mechanism of catabolite repression in the bgl operon of Escherichia coli: involvement of the anti-terminator BglG, CRP-cAMP and EIIAGlc in mediating glucose effect downstream of transcription initiationGENES TO CELLS, Issue 4 2000Abhilasha Gulati Background Expression of the bgl operon of Escherichia coli, involved in the regulated uptake and utilization of aromatic ,-glucosides, is extremely sensitive to the presence of glucose in the growth medium. We have analysed the mechanism by which glucose exerts its inhibitory effect on bgl expression. Results Our studies show that initiation of transcription from the bgl promoter is only marginally sensitive to glucose. Instead, glucose exerts a more significant inhibition on the elongation of transcription beyond the rho-independent terminator present within the leader sequence. Transcriptional analyses using plasmids that carry mutations in bglG or within the terminator, suggest that the target for glucose-mediated repression is the anti-terminator protein, BglG. Introduction of multiple copies of bglG or the presence of mutations that inhibit its phosphorylation by Enzyme IIBgl (BglF), result in loss of glucose repression. Studies using crp, cya and crr strains show that both CRP-cAMP and the Enzyme IIAGlc (EIIAGlc) are involved in the regulation. Although transcription initiation is normal in a crp, cya double mutant, no detectable transcription is seen downstream of the terminator, which is restored by a mutation within the terminator. Transcription past the terminator is also partly restored by the addition of exogenous cAMP to glucose-grown cultures of a crp+ strain. Glucose repression is lost in the crr mutant strain. Conclusions The results summarized above indicate that glucose repression in the bgl operon is mediated at the level of transcription anti-termination, and glucose affects the activity of BglG by altering its phosphorylation by BglF. The CRP-cAMP complex is also involved in this regulation. The results using the crr mutant suggest a negative role for EIIAGlc in the catabolite repression of the bgl genes. [source] Sfrp1, Sfrp2, and Sfrp5 regulate the Wnt/,-catenin and the planar cell polarity pathways during early trunk formation in mouseGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2008Wataru Satoh The secreted frizzled-related protein gene family encodes proteins that regulate Wnt signaling. Msx1 in situ hybridization of 9.5 days post coitus mouse embryos showing normal neural tube development in an Sfrp1; Sfrp2 double mutant (left) but severe neural tube defects in a Looptail (Lp/+); Sfrp1; Sfrp2 triple mutant (right). These findings suggest that Sfrps regulate the Wnt planar cell polarity pathway. See Satoh et al. in this issue. [source] Lipophilic regulator of a developmental switch in Caenorhabditis elegansAGING CELL, Issue 6 2004Matthew S. Gill In Caenorhabditis elegans, the decision to develop into a reproductive adult or arrest as a dauer larva is influenced by multiple pathways including insulin-like and transforming growth factor , (TGF,)-like signalling pathways. It has been proposed that lipophilic hormones act downstream of these pathways to regulate dauer formation. One likely target for such a hormone is DAF-12, an orphan nuclear hormone receptor that mediates these developmental decisions and also influences adult lifespan. In order to find lipophilic hormones we have generated lipophilic extracts from mass cultures of C. elegans and shown that they rescue the dauer constitutive phenotype of class 1 daf-2 insulin signalling mutants and the TGF, signalling mutant daf-7. These extracts are also able to rescue the lethal dauer phenotype of daf-9 mutants, which lack a P450 steroid hydroxylase thought to be involved in the synthesis of the DAF-12 ligand; extracts, however, have no effect on a DAF-12 ligand binding domain mutant that is predicted to be ligand insensitive. The production of this hormone appears to be DAF-9 dependent as extracts from a daf-9;daf-12 double mutant do not exhibit this activity. Preliminary fractionation of the lipophilic extracts shows that the activity is hydrophobic with some polar properties, consistent with a small lipophilic hormone. We propose that the dauer rescuing activity is a hormone synthesized by DAF-9 that acts through DAF-12. [source] WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repairAGING CELL, Issue 4 2003Lishan Chen Summary Werner syndrome (WS) predisposes patients to cancer and premature aging, owing to mutations in WRN. The WRN protein is a RECQ-like helicase and is thought to participate in DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) or homologous recombination (HR). It has been previously shown that non-homologous DNA ends develop extensive deletions during repair in WS cells, and that this WS phenotype was complemented by wild-type (wt) WRN. WRN possesses both 3, , 5, exonuclease and 3, , 5, helicase activities. To determine the relative contributions of each of these distinct enzymatic activities to DSB repair, we examined NHEJ and HR in WS cells (WRN,/,) complemented with either wtWRN, exonuclease-defective WRN (E,), helicase-defective WRN (H,) or exonuclease/helicase-defective WRN (E,H,). The single E, and H, mutants each partially complemented the NHEJ abnormality of WRN,/, cells. Strikingly, the E,H, double mutant complemented the WS deficiency nearly as efficiently as did wtWRN. Similarly, the double mutant complemented the moderate HR deficiency of WS cells nearly as well as did wtWRN, whereas the E, and H, single mutants increased HR to levels higher than those restored by either E,H, or wtWRN. These results suggest that balanced exonuclease and helicase activities of WRN are required for optimal HR. Moreover, WRN appears to play a structural role, independent of its enzymatic activities, in optimizing HR and efficient NHEJ repair. Another human RECQ helicase, BLM, suppressed HR but had little or no effect on NHEJ, suggesting that mammalian RECQ helicases have distinct functions that can finely regulate recombination events. [source] CLONING AND SEQUENCING OF THE ,-AMYLASE GENE FROM BACILLUS SUBTILIS US116 STRAIN ENCODING AN ENZYME CLOSELY IDENTICAL TO THAT FROM BACILLUS AMYLOLIQUEFACIENS BUT DISTINCT IN THERMAL STABILITYJOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2010EZZEDINE BEN MESSAOUD ABSTRACT The gene encoding for the ,-amylase AMYUS116 was cloned and sequenced. The amino acid sequence of AMYUS116 exhibited an almost perfect homology with the ,-amylase BACAAM, excluding the residues N205 and N217 of AMYUS116 that were changed to H205 and I217 into BACAAM. Three mutant derivatives from AMYUS116 (N205H, N217I and N205H/N217I) were created by site-directed mutagenesis and their physicochemical and kinetic properties were compared with those of the wild-type enzymes. Therefore, the undertaken amylases mainly generated maltohexaose from starch and had radically the same kinetic parameters and optimum pH and temperature. They, however, were significantly distinct in thermal stability; AMYUS116 was more thermosensible as its half-life time at 80C was 13 min, while those of BACAAM and the double mutant were likewise 38 min. The single-mutant amylases exhibited an identically intermediate thermal stability as their half-life times at 80C were roughly 22 min. PRACTICAL APPLICATIONS Of particular interest to the current search is that the different thermal stability between AMYUS116 and BACAAM can lead to novel findings pertaining to protein stability, which can bring about new strategies for protein engineering. Basically, the comparative study of closely related amylases and the protein engineering of already existing ones are certainly important because they offer opportunities to understand the structure,function relationships of these biocatalysts. [source] Simultaneous inactivation of the wprA and dltB genes of Bacillus subtilis reduces the yield of ,-amylaseLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2002K. Stephenson Aims:,In Gram-positive bacteria, signal peptide-bearing secretory proteins are translocated through the cytoplasmic membrane and fold into their native conformation on the outside of the cell. The products of the Bacillus subtilis wprA and dltB genes separately influence post-translocational stages of the secretion process by mediating proteolytic degradation and folding of secretory proteins. Inactivation of either wprA or dltB in B. subtilis increases the yield of secretory proteins released into the culture medium in an intact and biologically active conformation. The aim of this work was to study the combined influence of these genes. Methods and Results:,A wprA/dltB double mutant was constructed, but did not have an additive effect on secretion and caused a significant reduction in the yield of ,-amylase. Conclusions and Significance:,The activities of the wprA gene and the dlt operon interact in a negative way to influence the growth cycle and protein secretion. The mechanism by which this may occur, and its potential significance for the secretion of native and non-native proteins from B. subtilis and related bacteria, is discussed. [source] Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thalianaMOLECULAR MICROBIOLOGY, Issue 2 2004Patrice Hamel Summary Most cellular ATP is produced within the mitochondria from ADP and Pi which are delivered across the inner-membrane by specific nuclearly encoded polytopic carriers. In Saccharomyces cerevisiae, some of these carriers and in particular the ADP/ATP carrier, are represented by several related isoforms that are distinct in their pattern of expression. Until now, only one mitochondrial Pi carrier (mPic) form, encoded by the MIR1 gene in S. cerevisiae, has been described. Here we show that the gene product encoded by the YER053C ORF also participates in the delivery of phosphate to the mitochondria. We have called this gene PIC2 for Pi carrier isoform 2. Overexpression of PIC2 compensates for the mitochondrial defect of the double mutant ,mir1 ,pic2 and restores phosphate transport activity in mitochondria swelling experiments. The existence of two isoforms of mPic does not seem to be restricted to S. cerevisiae as two Arabidopsis thaliana cDNAs encoding two different mPic-like proteins are also able to complement the double mutant ,mir1 ,pic2. Finally, we demonstrate that Pic2p is a mitochondrial protein and that its steady state level increases at high temperature. We propose that Pic2p is a minor form of mPic which plays a role under specific stress conditions. [source] A role for the Escherichia coli H-NS-like protein StpA in OmpF porin expression through modulation of micF RNA stabilityMOLECULAR MICROBIOLOGY, Issue 1 2000Padraig Deighan When a wild-type strain of Escherichia coli and its stpA, hns and stpA hns mutant derivatives were compared by two-dimensional protein gel electrophoresis, the levels of expression of several proteins were found to vary. One of these was identified as the outer membrane porin protein, OmpF. In the stpA hns double mutant, the level of OmpF was downregulated dramatically, whereas in hns or stpA single mutants, it was affected only slightly. Transcription from the ompF promoter was reduced by 64% in the double mutant; however, the level of ompF mRNA was reduced by 96%. This post-transcriptional expression was found to result from a strong reduction in the half-life of ompF message in the double mutant. The micF antisense RNA was shown to be involved in OmpF regulation by StpA using a strain deleted for micF. Moreover, micF antisense RNA accumulated considerably in an stpA hns background. Transcriptional data from a micF,lacZ fusion and measurements of micF RNA half-life confirmed that this was caused by transcriptional derepression of micF as a result of the hns lesion and increased micF RNA stability due to the absence of StpA (a known RNA chaperone). These data suggest a novel facet to the regulation of OmpF expression, namely destabilization of micF RNA by StpA. [source] The Heterotrimeric G-protein Complex Modulates Light Sensitivity in Arabidopsis thaliana Seed GerminationPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2009Javier F. Botto Release of dormancy and induction of seed germination are complex traits finely regulated by hormonal signals and environmental cues such as temperature and light. The Red (R):Far-Red (FR) phytochrome photoreceptors mediate light regulation of seed germination. We investigated the possible involvement of heterotrimeric G-protein complex in the phytochrome signaling pathways of Arabidopsis thaliana seed germination. Germination rates of null mutants of the alpha (G,) and beta (G,) subunits of the G-protein (Atgpa1-4 and agb1-2, respectively) and the double mutant (agb1-2/gpa1-4) are lower than the wildtype (WT) under continuous or pulsed light. The G, and G, subunits play a role in seed germination under hourly pulses of R lower than 0.1 ,mol m,2 s,1 whereas the G, subunit plays a role in higher R fluences. The germination of double mutants of G-protein subunits with phyA-211 and phyB-9 suggests that AtGPA1 seems to act as a positive regulator of phyA and probably phyB signaling pathways, while the role of AGB1 is ambiguous. The imbibition of seeds at 4°C and 35°C alters the R and FR light responsiveness of WT and G-protein mutants to a similar magnitude. Thus, G, and G, subunits of the heterotrimeric G-protein complex modulate light induction of seed germination by phytochromes and are dispensable for the control of dormancy by low and high temperatures prior to irradiation. We discuss the possible indirect role of the G-protein complex on the phytochrome-regulated germination through hormonal signaling pathways. [source] Interaction of the Halobacterial Transducer to a Halorhodopsin Mutant Engineered so as to Bind the Transducer: Cl, Circulation Within the Extracellular Channel,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2007Chisa Hasegawa An alkali-halophilic archaeum, Natronomonas pharaonis, contains two rhodopsins that are halorhodopsin (phR), a light-driven inward Cl, pump and phoborhodopsin (ppR), the receptor of negative phototaxis functioning by forming a signaling complex with a transducer, pHtrII (Sudo Y. et al., J. Mol. Biol. 357 [2006] 1274). Previously, we reported that the phR double mutant, P240T/F250YphR, can bind with pHtrII. This mutant itself can transport Cl,, while the net transport was stopped upon formation of the complex. The flash-photolysis data were analyzed by a scheme in which phR,P1,P2,P3,P4,phR. The P3 of the wild-type and the double mutant contained two components, X- and O-intermediates. After the complex formation, however, the P3 of the double mutant lacked the X-intermediate. These observations imply that the X-intermediate (probably the N-intermediate) is the state having Cl, in the cytoplasmic binding site and that the complex undergoes an extracellular Cl, circulation because of the inhibition of formation of the X-intermediate. [source] Uncoupling brassinosteroid levels and de-etiolation in peaPHYSIOLOGIA PLANTARUM, Issue 2 2002Gregory M. Symons The suggestion that brassinosteroids (BRs) have a negative regulatory role in de-etiolation is based largely on correlative evidence, which includes the de-etiolated phenotypes of, and increased expression of light-regulated genes in, dark-grown mutants defective in BR biosynthesis or response. However, we have obtained the first direct evidence which shows that endogenous BR levels in light-grown pea seedlings are increased, not decreased, in comparison with those grown in the dark. Similarly, we found no evidence of a decrease in castasterone (CS) levels in seedlings that were transferred from the dark to the light for 24 h. Furthermore, CS levels in the constitutively de-etiolated lip1 mutant are similar to those in wild-type plants, and are not reduced as is the case in the BR-deficient lkb plants. Unlike lip1, the pea BR-deficient mutants lk and lkb are not de-etiolated at the morphological or molecular level, as they exhibit neither a de-etiolated phenotype or altered expression of light-regulated genes when grown in the dark. Similarly, dark-grown WT plants treated with the BR biosynthesis inhibitor, Brz, do not exhibit a de-etiolated phenotype. In addition, analysis of the lip1lkb double mutant revealed an additive phenotype indicative of the two genes acting in independent pathways. Together these results strongly suggest that BR levels do not play a negative-regulatory role in de-etiolation in pea. [source] Enzymatic and structural analysis of the I47A mutation contributing to the reduced susceptibility to HIV protease inhibitor lopinavirPROTEIN SCIENCE, Issue 9 2008Klára Grantz, ková Abstract Lopinavir (LPV) is a second-generation HIV protease inhibitor (PI) designed to overcome resistance development in patients undergoing long-term antiviral therapy. The mutation of isoleucine at position 47 of the HIV protease (PR) to alanine is associated with a high level of resistance to LPV. In this study, we show that recombinant PR containing a single I47A substitution has the inhibition constant (Ki) value for lopinavir by two orders of magnitude higher than for the wild-type PR. The addition of the I47A substitution to the background of a multiply mutated PR species from an AIDS patient showed a three-order-of-magnitude increase in Ki in vitro relative to the patient PR without the I47A mutation. The crystal structure of I47A PR in complex with LPV showed the loss of van der Waals interactions in the S2/S2, subsites. This is caused by the loss of three side-chain methyl groups due to the I47A substitution and by structural changes in the A47 main chain that lead to structural changes in the flap antiparallel ,-strand. Furthermore, we analyzed possible interaction of the I47A mutation with secondary mutations V32I and I54V. We show that both mutations in combination with I47A synergistically increase the relative resistance to LPV in vitro. The crystal structure of the I47A/I54V PR double mutant in complex with LPV shows that the I54V mutation leads to a compaction of the flap, and molecular modeling suggests that the introduction of the I54V mutation indirectly affects the strain of the bound inhibitor in the PR binding cleft. [source] Combinatorial engineering to enhance thermostability of amylosucrasePROTEIN SCIENCE, Issue 6 2008Stéphane Emond Abstract Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50°C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B, domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50°C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30°C. [source] NMR and SAXS characterization of the denatured state of the chemotactic protein Che Y: Implications for protein folding initiationPROTEIN SCIENCE, Issue 6 2001Pascal Garcia Abstract The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X-ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that ,25% of the residues are involved in residual structures. Conformational shifts of the ,-protons, heteronuclear 15N-{1H} NOEs and 15N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior. According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein. The first of these subdomains, spanning residues 1,70, is shown here to exhibit a restricted mobility as compared to the rest of the protein. Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone 1H chemical shifts were compared to those in the intact protein under identical denaturing conditions. For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule. The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state. [source] Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in riceTHE PLANT JOURNAL, Issue 1 2009Yutaka Sato Summary Yellowing, which is related to the degradation of chlorophyll and chlorophyll,protein complexes, is a notable phenomenon during leaf senescence. NON-YELLOW COLORING1 (NYC1) in rice encodes a membrane-localized short-chain dehydrogenase/reductase (SDR) that is thought to represent a chlorophyll b reductase necessary for catalyzing the first step of chlorophyll b degradation. Analysis of the nyc1 mutant, which shows the stay-green phenotype, revealed that chlorophyll b degradation is required for the degradation of light-harvesting complex II and thylakoid grana in leaf senescence. Phylogenetic analysis further revealed the existence of NYC1-LIKE (NOL) as the most closely related protein to NYC1. In the present paper, the nol mutant in rice was also found to show a stay-green phenotype very similar to that of the nyc1 mutant, i.e. the degradation of chlorophyll b was severely inhibited and light-harvesting complex II was selectively retained during senescence, resulting in the retention of thylakoid grana even at a late stage of senescence. The nyc1 nol double mutant did not show prominent enhancement of inhibition of chlorophyll degradation. NOL was localized on the stromal side of the thylakoid membrane despite the lack of a transmembrane domain. Immunoprecipitation analysis revealed that NOL and NYC1 interact physically in vitro. These observations suggest that NOL and NYC1 are co-localized in the thylakoid membrane and act in the form of a complex as a chlorophyll b reductase in rice. [source] The AT-hook-containing proteins SOB3/AHL29 and ESC/AHL27 are negative modulators of hypocotyl growth in ArabidopsisTHE PLANT JOURNAL, Issue 1 2008Ian H. Street Summary SOB3, which encodes a plant-specific AT-hook motif containing protein, was identified from an activation-tagging screen for suppressors of the long-hypocotyl phenotype of a weak phyB allele, phyB-4. sob3-D (suppressor of phyB-4#3 dominant) overexpressing seedlings have shorter hypocotyls, and as adults develop larger flowers and leaves, and are delayed in senescence compared with wild-type plants. At the nucleotide level, SOB3 is closely related to ESCAROLA (ESC), which was identified in an independent activation-tagging screen. ESC overexpression also suppresses the phyB-4 long-hypocotyl phenotype, and confers an adult morphology similar to sob3-D, suggesting similar functions. Analysis of transgenic plants harboring SOB3:SOB3-GUS or ESC:ESC-GUS translational fusions, driven by their endogenous promoter regions, showed GUS activity in the hypocotyl and vasculature tissue in light- and dark-grown seedlings. A loss-of-function SOB3 allele (sob3-4) was generated through an ethyl methanesulfonate intragenic suppressor screen of sob3-D phyB-4 plants, and this allele was combined with a predicted null allele, disrupting ESC (esc-8), to examine potential genetic interactions. The sob3-4 esc-8 double mutant had a long hypocotyl in multiple fluence rates of continuous white, far-red, red and blue light. sob3-4 esc-8 phyB-9 and sob3-4 esc-8 cry-103 triple mutants also had longer hypocotyls than photoreceptor single mutants. In contrast, the sob3-4 esc-8 phyA-211 triple mutant was the same length as phyA-211 single mutants. Taken together, these data indicate that SOB3 and ESC act redundantly to modulate hypocotyl growth inhibition in response to light. [source] Function of plastidial pyruvate kinases in seeds of Arabidopsis thaliana,THE PLANT JOURNAL, Issue 3 2007Sébastien Baud Summary Pyruvate kinase (PK) catalyses the irreversible synthesis of pyruvate and ATP, which are both used in multiple biochemical pathways. These compounds are essential for sustained fatty acid production in the plastids of maturing Arabidopsis embryos. Using a real-time quantitative reverse transcriptase (RT)-PCR approach, the three genes encoding putative plastidial PKs (PKps) in Arabidopsis, namely PKp1 (At3g22960), PKp2 (At5g52920) and PKp3 (At1g32440), were shown to be ubiquitously expressed. However, only PKp1 and PKp2 exhibited significant expression in maturing seeds. The activity of PKp1 and PKp2 promoters was consistent with this pattern, and the study of the PKp1:GFP and PKp2:GFP fusion proteins confirmed the plastidial localization of these enzymes. To further investigate the function of these two PKp isoforms in seeds comprehensive functional analyses were carried out, including the cytological, biochemical and molecular characterization of two pkp1 and two pkp2 alleles, together with a pkp1pkp2 double mutant. The results obtained outlined the importance of these PKps for fatty acid synthesis and embryo development. Mutant seeds were depleted of oil, their fatty acid content was drastically modified, embryo elongation was retarded and, finally, seed germination was also affected. Together, these results provide interesting insights concerning the carbon fluxes leading to oil synthesis in maturing Arabidopsis seeds. The regulation of this metabolic network by the WRINKLED1 transcription factor is discussed, and emphasizes the role of plastidial metabolism and the importance of its tight regulation. [source] ABA-Hypersensitive Germination1 encodes a protein phosphatase 2C, an essential component of abscisic acid signaling in Arabidopsis seedTHE PLANT JOURNAL, Issue 6 2007Noriyuki Nishimura Summary The phytohormone abscisic acid (ABA) regulates physiologically important stress and developmental responses in plants. To reveal the mechanism of response to ABA, we isolated several novel ABA-hypersensitive Arabidopsis thaliana mutants, named ahg (ABA- hypersensitive germination). ahg1-1 mutants showed hypersensitivity to ABA, NaCl, KCl, mannitol, glucose and sucrose during germination and post-germination growth, but did not display any significant phenotypes in adult plants. ahg1-1 seeds accumulated slightly more ABA before stratification and showed increased seed dormancy. Map-based cloning of AHG1 revealed that ahg1-1 has a nonsense mutation in a gene encoding a novel protein phosphatase 2C (PP2C). We previously showed that the ahg3-1 mutant has a point mutation in the AtPP2CA gene, which encodes another PP2C that has a major role in the ABA response in seeds (Yoshida et al., 2006b). The levels of AHG1 mRNA were higher in dry seeds and increased during late seed maturation , an expression pattern similar to that of ABI5. Transcriptome analysis revealed that, in ABA-treated germinating seeds, many seed-specific genes and ABA-inducible genes were highly expressed in ahg1-1 and ahg3-1 mutants compared with the wild-type. Detailed analysis suggested differences between the functions of AHG1 and AHG3. Dozens of genes were expressed more strongly in the ahg1-1 mutant than in ahg3-1. Promoter,GUS analyses demonstrated both overlapping and distinct expression patterns in seed. In addition, the ahg1-1 ahg3-1 double mutant was more hypersensitive than either monogenic mutant. These results suggest that AHG1 has specific functions in seed development and germination, shared partly with AHG3. [source] Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutantsTHE PLANT JOURNAL, Issue 6 2007Dong-Yul Sung Summary A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA -like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, , -glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5. [source] STY1 regulates auxin homeostasis and affects apical,basal patterning of the Arabidopsis gynoeciumTHE PLANT JOURNAL, Issue 1 2006Joel J. Sohlberg Summary Gynoecia of the Arabidopsis mutant sty1-1 display abnormal style morphology and altered vascular patterning. These phenotypes, which are enhanced in the sty1-1 sty2-1 double mutant, suggest that auxin homeostasis or signalling might be affected by mutations in STY1 and STY2, both members of the SHI gene family. Chemical inhibition of polar auxin transport (PAT) severely affects the apical,basal patterning of the gynoecium, as do mutations in the auxin transport/signalling genes PIN1, PID and ETT. Here we show that the apical,basal patterning of sty1-1 and sty1-1 sty2-1 gynoecia is hypersensitive to reductions in PAT, and that sty1-1 enhances the PAT inhibition-like phenotypes of pin1-5, pid-8 and ett-1 gynoecia. Furthermore, we show that STY1 activates transcription of the flavin monooxygenase-encoding gene THREAD/YUCCA4, involved in auxin biosynthesis, and that changes in expression of STY1 and related genes lead to altered auxin homeostasis. Our results suggest that STY1 and related genes promote normal development of the style and affect apical,basal patterning of the gynoecium through regulation of auxin homeostasis. [source] An Arabidopsis porB porC double mutant lacking light-dependent NADPH:protochlorophyllide oxidoreductases B and C is highly chlorophyll-deficient and developmentally arrestedTHE PLANT JOURNAL, Issue 2 2003Genevičve Frick Summary A key reaction in the biosynthesis of chlorophylls (Chls) a and b from cyanobacteria through higher plants is the strictly light-dependent reduction of protochlorophyllide (Pchlide) a to chlorophyllide (Chlide) a. Angiosperms, unlike other photosynthetic organisms, rely exclusively upon this mechanism to reduce Pchlide and hence require light to green. In Arabidopsis, light-dependent Pchlide reduction is mediated by three structurally related but differentially regulated NADPH:Pchlide oxidoreductases, denoted as PORA, PORB, and PORC. The PORA and PORB genes, but not PORC, are strongly expressed early in seedling development. In contrast, expression of PORB and PORC, but not PORA, is observed in older seedlings and adult plants. We have tested the hypothesis that PORB and PORC govern light-dependent Chl biosynthesis throughout most of the plant development by identifying porB and porC mutants of Arabidopsis, the first higher plant por mutants characterized. The porB-1 and porC-1 mutants lack the respective POR transcripts and specific POR isoforms because of the interruption of the corresponding genes by a derivative of the maize Dissociation (Ds) transposable element. Single por mutants, grown photoperiodically, display no obvious phenotypes at the whole plant or chloroplast ultrastructural levels, although the porB-1 mutant has less extensive etioplast inner membranes. However, a light-grown porB-1 porC-1 double mutant develops a seedling-lethal xantha phenotype at the cotyledon stage, contains only small amounts of Chl a, and possesses chloroplasts with mostly unstacked thylakoid membranes. PORB and PORC thus seem to play redundant roles in maintaining light-dependent Chl biosynthesis in green plants, and are together essential for growth and development. [source] Double mutation cpSRP43,/cpSRP54, is necessary to abolish the cpSRP pathway required for thylakoid targeting of the light-harvesting chlorophyll proteinsTHE PLANT JOURNAL, Issue 5 2002Claire Hutin Summary Biochemical and genetic studies have established that the light-harvesting chlorophyll proteins (LHCPs) of the photosystems use the cpSRP (chloroplast signal recognition particle) pathway for their targeting to thylakoids. Previous analyses of single cpSRP mutants, chaos and ffc, deficient in cpSRP43 and cpSRP54, respectively, have revealed that half of the LHCPs are still integrated into the thylakoid membranes. Surprisingly, the effects of both mutations are additive in the double mutant ffc/chaos described here. This mutant has pale yellow leaves at all stages of growth and drastically reduced levels of all the LHCPs except Lhcb 4. Although the chloroplasts have a normal shape, the thylakoid structure is affected by the mutation, probably as a consequence of reduction of all the LHCPs. ELIPs (early light-inducible proteins), nuclear-encoded proteins related to the LHCP family and inducible by light stress, were also drastically reduced in the double mutant. However, proteins targeted by other chloroplastic targeting pathways (,pH, Sec and spontaneous pathways) accumulated to similar levels in the wild-type and the double mutant. Therefore, the near total loss of LHCPs and ELIPs in the double mutant suggests that cpSRP is the predominant, if not exclusive, targeting pathway for these proteins. Phenotypic analysis of the double mutant, compared to the single mutants, suggests that the cpSRP subunits cpSRP43 and cpSRP54 contribute to antenna targeting in an independent but additive way. [source] Mosquito NADPH-cytochrome P450 oxidoreductase: kinetics and role of phenylalanine amino acid substitutions at leu86 and leu219 in CYP6AA3-mediated deltamethrin metabolismARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2010Songklod Sarapusit Abstract The NADPH-cytochrome P450 oxidoreductase (CYPOR) enzyme is a membrane-bound protein and contains both FAD and FMN cofactors. The enzyme transfers two electrons, one at a time, from NADPH to cytochrome P450 enzymes to function in the enzymatic reactions. We previously expressed in Escherichia coli the membrane-bound CYPOR (flAnCYPOR) from Anopheles minimus mosquito. We demonstrated the ability of flAnCYPOR to support the An. minimus CYP6AA3 enzyme activity in deltamethrin degradation in vitro. The present study revealed that the flAnCYPOR purified enzyme, analyzed by a fluorometric method, readily lost its flavin cofactors. When supplemented with exogenous flavin cofactors, the activity of flAnCYPOR-mediated cytochrome c reduction was increased. Mutant enzymes containing phenylalanine substitutions at leucine residues 86 and 219 were constructed and found to increase retention of FMN cofactor in the flAnCYPOR enzymes. Kinetic study by measuring cytochrome c,reducing activity indicated that the wild-type and mutant flAnCYPORs followed a non-classical two-site Ping-Pong mechanism, similar to rat CYPOR. The single mutant (L86F or L219F) and double mutant (L86F/L219F) flAnCYPOR enzymes, upon reconstitution with the An. minimus cytochrome P450 CYP6AA3 and a NADPH-regenerating system, increased CYP6AA3-mediated deltamethrin degradation compared to the wild-type flAnCYPOR enzyme. The increased enzyme activity could illustrate a more efficient electron transfer of AnCYPOR to CYP6AA3 cytochrome P450 enzyme. Addition of extra flavin cofactors could increase CYP6AA3-mediated activity supported by wild-type and mutant flAnCYPOR enzymes. Thus, both leucine to phenylalanine substitutions are essential for flAnCYPOR enzyme in supporting CYP6AA3-mediated metabolism. © 2010 Wiley Periodicals, Inc. [source] | ||||||||||||||||||||||||||||||||||