scholarly journals Herbicide-resistant forms of Arabidopsis thaliana acetohydroxyacid synthase: characterization of the catalytic properties and sensitivity to inhibitors of four defined mutants

1998 ◽  
Vol 333 (3) ◽  
pp. 765-777 ◽  
Author(s):  
Alan K. CHANG ◽  
Ronald G. DUGGLEBY
Keyword(s):  
Arabidopsis Thaliana ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Acetohydroxyacid Synthase ◽  
Similar Degree ◽  
Mutant Form ◽  
Wild Type ◽  
Ph Optimum ◽  
Branched Chain ◽  
Imidazolinone Herbicides

Acetohydroxyacid synthase (AHAS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides. Four mutants (A122V, W574S, W574L and S653N) of the AHAS gene from Arabidopsis thaliana were constructed, expressed in Escherichia coli, and the enzymes were purified. Each mutant form and wild-type was characterized with respect to its catalytic properties and sensitivity to nine herbicides. Each enzyme had a pH optimum near 7.5. The specific activity varied from 13% (A122V) to 131% (W574L) of the wild-type and the Km for pyruvate of the mutants was similar to the wild-type, except for W574L where it was five-fold higher. The activation by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined. A122V showed reduced affinity for all three cofactors, whereas S653N bound FAD more strongly than wild-type AHAS. Six sulphonylurea herbicides inhibited A122V to a similar degree as the wild-type but S653N showed a somewhat greater reduction in sensitivity to these compounds. In contrast, the W574 mutants were insensitive to these sulphonylureas, with increases in the Kiapp (apparent inhibition constant) of several hundred fold. All four mutants were resistant to three imidazolinone herbicides with decreases in sensitivity ranging from 100-fold to more than 1000-fold.

Stimulation of Escherichia coli adenylate cyclase by lactose in strains carrying mutations in lactose permease

1981 ◽  
Vol 1 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Alan Peterkofsky ◽  
Celia Gazdar
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Specific Activity ◽  
Inhibitory Effect ◽  
Lactose Permease ◽  
Mutant Form ◽  
Wild Type ◽  
Camp Phosphodiesterase ◽  
Intact Cells

When a wild-type strain of Escherichia coli contains lactose permease, the accumulation of cyclic AMP (cAMP) by intact cells is inhibited by lactose. This inhibitory effect of lactose is observed in a strain with a mutant cAMP phosphodiesterase and therefore involves a regulation of adenylate cyctase activity. Some E. coli strains carrying mutations in lactose permease show an effect opposite to that of the wild-type strain; the accumulation of cAMP by intact cells is stimulated by lactose, but only when the mutant permease is present. Insertion of lactose permease into the membrane of ceils can produce a change in the specific activity of adenylate cycIase; induction of the wild-type transporter is correlated with a decrease in the specific activity, while implantation of a mutant form of lactose permease can lead to an increase in the specific activity. From these data, it is suggested that the state of the lactose transporter in the cell membrane influences the activity of adenytate cyclase.

scholarly journals Site-directed mutation of a laccase from Thermus thermophilus: Effect on the activity profile

2012 ◽  
Vol 64 (4) ◽  
pp. 1515-1522 ◽  
Author(s):  
Xin Liu ◽  
Meng Tian ◽  
Kewu Liu
Keyword(s):  
Catalytic Properties ◽  
Specific Activity ◽  
Affinity Purification ◽  
Thermus Thermophilus ◽  
Copper Ion ◽  
Substrate Affinity ◽  
Wild Type ◽  
Directed Mutation ◽  
Nickel Affinity Purification ◽  
A Site

A site-directed mutant R453T of a laccase from Thermus thermophilus HB27 (Tth-laccase) was constructed in order to investigate the effect on laccase catalytic properties. The mutated gene was cloned and overexpressed in Escherichia coli. Nickel-affinity purification was achieved and followed by copper ion incorporation. The mature mutated enzyme was quantitatively equal to the wild type. A photometric assay based on the oxidation of the substrate 2,2-azino-bis-(3- ethylbenzthiazoline-6-sulfonate) (ABTS) was employed in comparison with the wild-type Tth-laccase on catalytic properties. The R453T mutant exhibited improvement in substrate affinity and specific activity at room temperature, whereas those parameters were not significantly influenced when the temperature increased up to 65?C or higher. The mutant had better catalytic activity than that of the wild type at acidic pH. Investigated by circular dichroism spectroscopy, the mutant Tth-laccase displayed similar profiles at low and high temperatures.

An Ala205Val Substitution in Acetohydroxyacid Synthase of Eastern Black Nightshade (Solanum ptychanthum) Reduces Sensitivity to Herbicides and Feedback Inhibition

Weed Science ◽  
2007 ◽  
Vol 55 (6) ◽  
pp. 558-565 ◽  
Author(s):  
Jamshid Ashigh ◽  
François J. Tardif
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Acetohydroxyacid Synthase ◽  
Susceptible Population ◽  
Eastern Black Nightshade ◽  
Black Nightshade ◽  
Branched Chain Amino Acid ◽  
Resistant Population ◽  
Branched Chain

Twelve populations of eastern black nightshade from different locations in Ontario are resistant to imazethapyr. This study aimed at determining the molecular basis of resistance in these populations and the activity of the resistant acetohydroxyacid synthase (AHAS) enzyme compared to that of the sensitive AHAS in response to different herbicides and branched-chain amino acid concentration. The results of partialAHASsequencing indicated that all resistant populations had a cytosine331to thymine substitution coding for an alanine205to valine substitution.In vitroAHAS enzyme assays of one resistant population showed that the specific activity of the resistant enzyme was 56% less than that of the susceptible enzyme. AHAS from the resistant population was 72-, 70-, and 64-fold less sensitive than that of the susceptible population to imazethapyr, imazamox, and primisulfuron, respectively. Furthermore, the resistant enzyme was less sensitive to feedback inhibition from branched-chain amino acids compared to the susceptible enzyme. Results confirmed that resistance in resistant populations of eastern black nightshade was conferred by target-site modification and that the Ala205Val substitution alters the kinetics and regulation of branched-chain amino acid biosynthesis.

scholarly journals Mutagenesis of Escherichia coli acetohydroxyacid synthase isoenzyme II and characterization of three herbicide-insensitive forms

1998 ◽  
Vol 335 (3) ◽  
pp. 653-661 ◽  
Author(s):  
Craig M. HILL ◽  
Ronald G. DUGGLEBY
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Molecular Model ◽  
Small Subunit ◽  
Acetohydroxyacid Synthase ◽  
Kinetic Properties ◽  
Wild Type ◽  
Thiamin Diphosphate ◽  
E Coli ◽  
Branched Chain

Sulphonylurea and imidazolinone herbicides act by inhibiting acetohydroxyacid synthase (AHAS; EC 4.1.3.18), the enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids. AHAS requires as cofactors thiamin diphosphate, a bivalent metal ion and, usually, FAD. Escherichia coli contains three isoenzymes and this study concerns isoenzyme II, the most herbicide-sensitive of the E. coli forms. A plasmid containing the large and small subunit genes of AHAS II was mutagenized using hydroxylamine and clones resistant to the sulphonylurea chlorimuron ethyl were selected. Three mutants were isolated; A26V, V99M and A108V. A26V has been described previously whereas the equivalent mutation of A108V has been reported in a herbicide-insensitive variant of yeast AHAS. The V99M mutation has not been discovered previously in AHAS from any source. The mutants were each over-expressed in E. coli, and the enzymes were purified to homogeneity. Some differences from wild type in the kinetic properties (kcat, Km and cofactor affinities) were observed, most notably a 28-fold decrease in the affinity for thiamin diphosphate of V99M. None of the mutants shows marked changes from the wild type in sensitivity to three imidazolinones, with the largest increase in the apparent inhibition constant being a factor of approximately 5. The A26V mutant is weakly resistant (6- to 20-fold) to six sulphonylureas, whereas stronger resistance is seen in V99M (20- to 250-fold) and A108V (35- to 420-fold). Resistance as a result of these mutations is consistent with a molecular model of the herbicide-binding site, which predicts that mutation of G249 might also confer herbicide insensitivity. Three G249 mutants were constructed, expressed and purified but all are inactive, apparently because they cannot bind FAD.

scholarly journals Effects of directed mutagenesis on conserved arginine residues in a human Class Alpha glutathione transferase

1991 ◽  
Vol 274 (2) ◽  
pp. 549-555 ◽  
Author(s):  
G Stenberg ◽  
P G Board ◽  
I Carlberg ◽  
B Mannervik
Keyword(s):  
Glutathione Transferase ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Cumene Hydroperoxide ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Conformational States ◽  
Arginine Residues ◽  
Increased Sensitivity ◽  
Inhibition Studies

Glutathione transferase (GST) epsilon (also known as GST2 or GST B1B1), the major Class Alpha GST in human liver has been subjected to oligonucleotide-directed site-specific mutagenesis. Four arginine residues, R13, R20, R69 and R187, of which all but R69 are strictly conserved through GST Classes Alpha, Mu and Pi have been replaced by Ala. The mutant enzymes have been expressed in Escherichia coli, purified by affinity chromatography and characterised. Compared with the wild-type enzyme, all mutant GSTs had altered catalytic properties. All mutants had decreased specific activity with 1-chloro-2,4-dinitrobenzene (CDNB). Mutants R13A, R69A and R187A also showed decreased activities with other substrates such as cumene hydroperoxide (CuOOH) and androstenedione. In contrast, mutant R20A had an increased peroxidase activity and an isomerase activity essentially the same as that of the wild-type GST. With the substrates used, kcat./Km values were decreased for all mutant GSTs. Increases in the [S0.5] values were most significant for glutathione (GSH), while values for CDNB and CuOOH were less markedly affected. Thus, various kinetic data indicate that the GSH affinity has been reduced by the mutations and that this loss of affinity is linked to the decreased specific activities. Inhibition studies showed an increased sensitivity towards S-hexyl-GSH; this was particularly marked for mutant R69A. Mutant R20A had a lowered [I50] value but, in contrast, also the highest [I80] value as compared with the wild-type enzyme. Towards bromosulphophthalein, mutants R20A and R69A had a markedly increased sensitivity, about 35-fold in comparison with the wild-type. The inhibition properties of mutant R187A were similar to those of the wild-type enzyme and the properties of mutant R13A were in between. The increased sensitivity to S-hexyl-GSH, in contrast with the decreased affinity for GSH, was suggested to be due to an altered distribution between conformational states of the enzyme induced by the mutations. The arginine residues in positions 13, 20 and 69 all seem to be important for the catalytic properties of GST. Further, the inhibition studies indicate a role of arginine residues in the stabilisation of conformational states of the enzyme.

scholarly journals Amino- and carboxy-terminal deletion mutants of Gs alpha are localized to the particulate fraction of transfected COS cells.

1992 ◽  
Vol 119 (3) ◽  
pp. 523-530 ◽  
Author(s):  
Y S Juhnn ◽  
T L Jones ◽  
A M Spiegel
Keyword(s):  
Amino Acid ◽  
Particulate Fraction ◽  
Structural Basis ◽  
Similar Degree ◽  
Mutant Form ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Amino Terminal ◽  
Gs Alpha ◽  
Mutant Forms

To elucidate the structural basis for membrane attachment of the alpha subunit of the stimulatory G protein (Gs alpha), mutant Gs alpha cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gs alpha with either 26 amino terminal residues deleted (delta 3-28) or with 59 amino terminal residues deleted (delta 1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (delta 385-394), 32 (delta 353-384), or 42 (delta 353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gs alpha lacking amino acid residues at both the amino and carboxy termini (delta 3-28)/(delta 353-384). Mutant and wild type forms of Gs alpha demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gs alpha to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gs alpha is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gs alpha membrane targeting remains to be elucidated.

scholarly journals Branched-Chain Amino Acids Are Required for the Survival and Virulence of Actinobacillus pleuropneumoniae in Swine

2009 ◽  
Vol 77 (11) ◽  
pp. 4925-4933 ◽  
Author(s):  
Sargurunathan Subashchandrabose ◽  
Rhiannon M. LeVeque ◽  
Trevor K. Wagner ◽  
Roy N. Kirkwood ◽  
Matti Kiupel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Antimicrobial Agents ◽  
Actinobacillus Pleuropneumoniae ◽  
Defined Medium ◽  
Acetohydroxyacid Synthase ◽  
Infection Model ◽  
Wild Type ◽  
Branched Chain

ABSTRACT In Actinobacillus pleuropneumoniae, which causes porcine pleuropneumonia, ilvI was identified as an in vivo-induced (ivi) gene and encodes the enzyme acetohydroxyacid synthase (AHAS) required for branched-chain amino acid (BCAA) biosynthesis. ilvI and 7 of 32 additional ivi promoters were upregulated in vitro when grown in chemically defined medium (CDM) lacking BCAA. Based on these observations, we hypothesized that BCAA would be found at limiting concentrations in pulmonary secretions and that A. pleuropneumoniae mutants unable to synthesize BCAA would be attenuated in a porcine infection model. Quantitation of free amino acids in porcine pulmonary epithelial lining fluid showed concentrations of BCAA ranging from 8 to 30 μmol/liter, which is 10 to 17% of the concentration in plasma. The expression of both ilvI and lrp, a global regulator that is required for ilvI expression, was strongly upregulated in CDM containing concentrations of BCAA similar to those found in pulmonary secretions. Deletion-disruption mutants of ilvI and lrp were both auxotrophic for BCAA in CDM and attenuated compared to wild-type A. pleuropneumoniae in competitive index experiments in a pig infection model. Wild-type A. pleuropneumoniae grew in CDM+BCAA but not in CDM−BCAA in the presence of sulfonylurea AHAS inhibitors. These results clearly demonstrate that BCAA availability is limited in the lungs and support the hypothesis that A. pleuropneumoniae, and potentially other pulmonary pathogens, uses limitation of BCAA as a cue to regulate the expression of genes required for survival and virulence. These results further suggest a potential role for AHAS inhibitors as antimicrobial agents against pulmonary pathogens.

Heterologous Expression and Functional Characterization of Catalytic Subunit of Rice Acetohydroxyacid Synthase

2019 ◽  
Vol 26 (3) ◽  
pp. 176-183
Author(s):  
Ghazaleh Arabzadeh ◽  
Azar Shahpiri
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Functional Characterization ◽  
Branched Chain Amino Acids ◽  
Biochemical Properties ◽  
Catalytic Subunit ◽  
Acetohydroxyacid Synthase ◽  
Branched Chain

Background: Acetohydroxyacid Synthase (AHAS) is the first enzyme in the biosynthesis pathway of the branched chain amino acids. AHAS is the common target site of five herbicide chemical groups: sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl-thiobenzoates, and sulfonyl-aminocarbonyl-triazolinone. </P><P> Objective: The purification of protein enabled us to study the physical and biochemical properties of the enzyme. In addition in vitro activity of this enzyme was tested in the presence of four different sulfonylureaherbicides and the feedback regulation of enzyme was analyzed in the presence of branched amino acids. Methods: The gene encoding catalytic subunit of rice AHAS (cOsAHAS) without part of the chloroplast transit sequence was cloned into the bacterial expression vector pET41a and heterologously expressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST).The soluble protein was purified using affinity chromatography. The measurement of GSTOsAHAS activity was performed under optimized conditions at present of branched-chain amino acids and sulfonylurea herbicides independently. Results: The optimum pH and temperature for GST-cOsAHAS activity was 8.0 and 37 °C, respectively. The specific activity and Km value of this enzyme toward pyruvate were 0.08 U/mg and 30 mM, respectively.GST-cOsAHAS was inhibited by herbicides tribenuron, sulfosulfuron, nicosulfuron and bensulfuron while the enzyme was insensitivieto end products. Conclusion: These results suggest that the recombinant form of GST-cOsAHAS is functionally active and carries the binding site for sulfynylurea herbicides. Furthermore, GST-cOsAHAS was insensitive to feedback inhibition by endproducts which indicates the existence of a regulator subunit in rice AHAS as previously has been described in other plant AHASs.

Tyrosine 39 of GH13 α-amylase from Thermococcus hydrothermalis contributes to its thermostability

Biologia ◽  
2010 ◽  
Vol 65 (3) ◽  
Author(s):  
Andrej Godány ◽  
Katarína Majzlová ◽  
Viera Horváthová ◽  
Barbora Vidová ◽  
Štefan Janeček
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Thermotoga Maritima ◽  
Amino Acid Sequences ◽  
Point Of View ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Ph Optimum ◽  
Tim Barrel

AbstractThe presented work is focused on the naturally thermostable α-amylase from the archaebacterium Thermococcus hydrothermalis. From the evolutionary point of view, the archaeal α-amylases are most closely related to plant α-amylases. In a wider sense, especially when the evolutionary trees are based on the less conserved part of their amino acid sequences (e.g. domain C succeeding the catalytic TIM-barrel), also the representatives of bacterial liquefying (Bacillus licheniformis) and saccharifying (Bacillus subtilis) α-amylases as well as the one from Thermotoga maritima should be included into the relatedness with the archaeal and plant α-amylases. Based on the bioinformatics analysis of the α-amylase from T. hydrothermalis, the position of tyrosine 39 (Y16 if the putative 23-residue long signal peptide is considered) was mutated to isoleucine (present in the α-amylase from T. maritima) by the in vitro mutagenesis. The biochemical characterization of the wild-type α-amylase and its Y39I mutant revealed that: (i) the specific activity of both enzymes was approximately equivalent (0.55 ± 0.13 U/mg for the wild-type and 0.52 ± 0.15 U/mg for the Y39I); (ii) the mutant exhibited decreased temperature optimum (from 85°C for the wild-type to 80°C for the Y39I); and (iii) the pH optimum remained the same (pH 5.5 for both enzymes). The remaining activity of the α-amylases was also tested by one-hour incubation at 80°C, 85°C, 90°C and 100°C. Since the wild-type α-amylase lost only 13% of its activity after one-hour incubation at the highest tested temperature (100°C), whereas 27% decrease was seen for the mutant Y39I under the same conditions, it is possible to conclude that the position of tyrosine 39 could contribute to the thermostability of the α-amylase from T. hydrothermalis.

Characterization of catalase activities in a root-colonizing isolate of Pseudomonas putida

1992 ◽  
Vol 38 (10) ◽  
pp. 1026-1032 ◽  
Author(s):  
J. Katsuwon ◽  
A. J. Anderson
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Growth Phase ◽  
Specific Activity ◽  
Active Oxygen Species ◽  
Stationary Growth Phase ◽  
Wild Type ◽  
Ph Optimum ◽  
Chloroform Methanol ◽  
Root Surfaces

Pseudomonas putida, a saprophytic root-colonizing bacterium, produces multiple forms of catalase. Catalase A, which increases in specific activity during growth phase and after treatment with H2O2, is located in the cytoplasm and is inhibited by 3-amino-1,2,4-triazole, EDTA, and cyanide, but not by chloroform–methanol treatment. Catalase B, which is induced by external H2O2 or during stationary phase of growth, is membrane associated and is inhibited by chloroform–methanol, EDTA, and cyanide, but not by aminotriazole. Catalase A has a broad pH optimum, from pH 6.0 to 11.0, with two peaks, at pH 8.0 and 11.0. Catalase B is most active at pH 5.0–11.0. Mutant J-1, generated by ethyl methanesulfonate mutagenesis, lacked catalase A activity in extracts of cells harvested throughout lag to early stationary growth phase in liquid medium. Catalase B was produced by J-1 in stationary phase. Exposure of J-1 to H2O2 caused the production of both catalase A and catalase B. Mutant J-1 was more susceptible to cell death than the wild type upon direct exposure to 2.5 mM H2O2 but survived this treatment after exposure to lower (0.3 mM), nonlethal doses of H2O2. The ability to adapt to H2O2 may be related to the behaviour of J-1 on roots where active oxygen species are produced by root surface enzymes. J-1 colonized root surfaces at wild-type levels and produced catalases A and B after exposure to root surfaces for 12 h. Key words: Pseudomonas putida, catalase, root colonization.

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