scholarly journals Aspartate-107 and leucine-109 facilitate efficient coupling of glutamine hydrolysis to CTP synthesis by Escherichia coli CTP synthase

2003 ◽  
Vol 369 (3) ◽  
pp. 497-507 ◽  
Author(s):  
Akshai IYENGAR ◽  
Stephen L. BEARNE
Keyword(s):  
Escherichia Coli ◽  
Normal Structure ◽  
Kinetic Properties ◽  
Wild Type ◽  
Nucleotide Binding Sites ◽  
Allosteric Effector ◽  
Close Proximity ◽  
Ctp Synthase ◽  
Dependent Activity ◽  
Glutaminase Activity

CTP synthase catalyses the ATP-dependent formation of CTP from UTP using either NH3 or l-glutamine as the nitrogen source. GTP is required as an allosteric effector to promote glutamine hydrolysis. In an attempt to identify nucleotide-binding sites, scanning alanine mutagenesis was conducted on a highly conserved region of amino acid sequence (residues 102—118) within the synthase domain of Escherichia coli CTP synthase. Mutant K102A CTP synthase exhibited wild-type activity with respect to NH3 and glutamine; however, the R105A, D107A, L109A and G110A enzymes exhibited wild-type NH3-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation. The E103A, R104A and H118A enzymes exhibited no glutamine-dependent activity and were only partially active with NH3. Although these observations were compatible with impaired activation by GTP, the apparent affinity of the D107A, L109A and G110A enzymes for GTP was reduced only 2—4-fold, suggesting that these residues do not play a significant role in GTP binding. In the presence of GTP, the kcat values for glutamine hydrolysis by the D107A and L109A enzymes were identical with that of wild-type CTP synthase. Overall, the kinetic properties of L109A CTP synthase were consistent with an uncoupling of glutamine hydrolysis from CTP formation that occurs because an NH3 tunnel has its normal structure altered or fails to form. L109F CTP synthase was prepared to block totally the putative NH3 tunnel; however, this enzyme's rate of glutamine-dependent CTP formation and glutaminase activity were both impaired. In addition, we observed that mutation of amino acids located between residues 102 and 118 in the synthase domain can affect the enzyme's glutaminase activity, suggesting that these residues interact with residues in the glutamine amide transfer domain because they are in close proximity or via a conformationally dependent signalling mechanism.

Evolving improved Synechococcus Rubisco functional expression in Escherichia coli

2008 ◽  
Vol 414 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Oliver Mueller-Cajar ◽  
Spencer M. Whitney
Keyword(s):  
Escherichia Coli ◽  
Large Subunit ◽  
Fold Increase ◽  
Functional Expression ◽  
Kinetic Properties ◽  
Selection System ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
E Coli ◽  
Enhanced Expression

The photosynthetic CO2-fixing enzyme Rubisco [ribulose-P2 (D-ribulose-1,5-bisphosphate) carboxylase/oxygenase] has long been a target for engineering kinetic improvements. Towards this goal we used an RDE (Rubisco-dependent Escherichia coli) selection system to evolve Synechococcus PCC6301 Form I Rubisco under different selection pressures. In the fastest growing colonies, the Rubisco L (large) subunit substitutions I174V, Q212L, M262T, F345L or F345I were repeatedly selected and shown to increase functional Rubisco expression 4- to 7-fold in the RDE and 5- to 17-fold when expressed in XL1-Blue E. coli. Introducing the F345I L-subunit substitution into Synechococcus PCC7002 Rubisco improved its functional expression 11-fold in XL1-Blue cells but could not elicit functional Arabidopsis Rubisco expression in the bacterium. The L subunit substitutions L161M and M169L were complementary in improving Rubisco yield 11-fold, whereas individually they improved yield ∼5-fold. In XL1-Blue cells, additional GroE chaperonin enhanced expression of the I174V, Q212L and M262T mutant Rubiscos but engendered little change in the yield of the more assembly-competent F345I or F345L mutants. In contrast, the Rubisco chaperone RbcX stimulated functional assembly of wild-type and mutant Rubiscos. The kinetic properties of the mutated Rubiscos varied with noticeable reductions in carboxylation and oxygenation efficiency accompanying the Q212L mutation and a 2-fold increase in Kribulose-P2 (KM for the substrate ribulose-P2) for the F345L mutant, which was contrary to the ∼30% reductions in Kribulose-P2 for the other mutants. These results confirm the RDE systems versatility for identifying mutations that improve functional Rubisco expression in E. coli and provide an impetus for developing the system to screen for kinetic improvements.

KINETICS OF THREONINE DEAMINASE OF ESCHERICHIA COLI K-12 AND A STREPTOMYCIN-DEPENDENT MUTANT

1967 ◽  
Vol 45 (1) ◽  
pp. 1-10 ◽  
Author(s):  
I. D. Desai ◽  
W. J. Polglase
Keyword(s):  
Escherichia Coli ◽  
Selective Advantage ◽  
Kinetic Properties ◽  
Wild Type ◽  
Threonine Deaminase ◽  
E Coli ◽  
Type K ◽  
Hyperbolic Curve ◽  
K 12 ◽  
Kinetics Of

The relation between threonine deaminase activity and threonine concentration in sonic extracts of wild-type and streptomycin-dependent Escherichia coli K-12 was found to follow a hyperbolic curve. A similar relationship was obtained between enzyme activity and pyridoxal concentration. However, when serine was used as substrate, the activity–concentration curve was sigmoid, suggesting that serine may be a weaker effector of allosteric transition than threonine. The kinetic properties of the (derepressed) threonine deaminase of streptomycin-dependent E. coli K-12 were found to be similar to those of the enzyme of the wild-type K-12.It is postulated that derepression of threonine deaminase in streptomycin-dependent E. coli K-12 provides a selective advantage which permits exponential growth of this mutant in the presence of L-valine, which is an excretory product of streptomycin-dependent microorganisms.

Inhibition of Escherichia coli CTP synthase by glutamate γ-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis

2001 ◽  
Vol 356 (1) ◽  
pp. 223-232 ◽  
Author(s):  
Stephen L. BEARNE ◽  
Omid HEKMAT ◽  
Jennifer E. MacDONNELL
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Specific Activity ◽  
High Specific Activity ◽  
Competitive Inhibitor ◽  
Enzymic Activity ◽  
Open Chain ◽  
Tetrahedral Intermediate ◽  
Allosteric Effector ◽  
Ctp Synthase

Cytidine 5′-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate γ-semialdehyde. Glutamate γ-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate γ-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (Kis 0.16±0.03mM; Kii 0.4±0.1mM) and a competitive inhibitor with respect to ammonia (Ki 0.39±0.06mM) in the presence of GTP at pH8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate γ-semialdehyde. Although glutamate γ-semialdehyde exists in solution primarily in its cyclic form, Δ1-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Δ1-pyrroline-5-carboxylate (l-proline, l-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate γ-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis.

A mutant of Escherichia coli citrate synthase that affects the allosteric equilibrium

1991 ◽  
Vol 69 (4) ◽  
pp. 232-238 ◽  
Author(s):  
Deborah H. Anderson ◽  
Lynda J. Donald ◽  
Mary V. Jacob ◽  
Harry W. Duckworth
Keyword(s):  
Escherichia Coli ◽  
Model Building ◽  
Citrate Synthase ◽  
Salt Bridge ◽  
Difference Spectrum ◽  
Site Directed Mutagenesis ◽  
Kinetic Properties ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
In The Wild

We describe a mutant of Escherichia coli citrate synthase, CS R319L, in which the arginine residue at position 319 of the sequence has been replaced by leucine. In this mutant, saturation by the substrate acetyl-CoA is changed from sigmoid (Hill parameter = 1.75 ± 0.2) to hyperbolic (Hill parameter = 1.0 ± 0.1) and dependence on the activator KCl is greatly reduced. Further mutations at this site and at position 343 (which model building predicts is close enough to allow a side-chain interaction with position 319) are also described. In the wild-type enzyme, the model suggests the possibility of a salt-bridge interaction between Arg-319 (located on the P helix in the small domain) and Glu-343 (in the Q helix in the same domain), but mutation of Glu-343 to Ala (CS E343A) produced a much smaller difference in the kinetic properties than the Arg-319 to Leu mutation did. Small changes in kinetic properties were also obtained with an Arg-319 → Glu (CS R319E) mutation. In CS R319L, oxaloacetate, the first substrate to bind, induces an ultraviolet difference spectrum which is obtained with wild-type enzyme only in the presence of KCl. To account for these observations we postulate that wild-type E. coli citrate synthase exists in two conformational states, T and R, which are equilibrium; T state binds NADH, the allosteric inhibitor, while R state binds substrates and can be converted to another substrate-binding state, R′, by KCl. In the CS R319L mutant, it is proposed that the T ↔ R equilibrium is shifted significantly towards R state, permitting an easier interaction with substrates in the absence of KCl. To account for the behaviour of enzymes mutated at amino acids 319 and 343, we propose that the allosteric transition between T and R states involves a subtle adjustment of the relative positions of the P and Q helices, which is affected by some of the mutations tested.Key words: citrate synthase, allostery, site-directed mutagenesis.

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.

Kinetic properties of dihydrofolate reductase from wild-type and mutant Plasmodium vivax expressed in Escherichia coli

2001 ◽  
Vol 113 (2) ◽  
pp. 241-249 ◽  
Author(s):  
Rachida Tahar ◽  
Philippe Eldin de Pécoulas ◽  
Leonardo K. Basco ◽  
Mohammed Chiadmi ◽  
André Mazabraud
Keyword(s):  
Escherichia Coli ◽  
Plasmodium Vivax ◽  
Dihydrofolate Reductase ◽  
Kinetic Properties ◽  
Wild Type

scholarly journals Site-directed mutagenesis of cysteine-195 in isocitrate lyase from Escherichia coli ML308

1995 ◽  
Vol 305 (1) ◽  
pp. 239-244 ◽  
Author(s):  
A G S Robertson ◽  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Isocitrate Lyase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Active Site Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Native Page ◽  
Mutant Proteins

Cysteine-195 was previously identified as a probable active site residue in isocitrate lyase (ICL) from Escherichia coli ML308 [Nimmo, Douglas, Kleanthous, Campbell and MacKintosh (1989) Biochem. J. 261, 431-435]. This residue was replaced with serine and alanine residues by site-directed mutagenesis. The mutated genes expressed proteins with low but finite ICL activity, which co-migrated with wild-type ICL on both SDS/ and native PAGE. The mutant proteins were purified and characterized. Fluorimetry and c.d. in both the near- and the far-u.v. regions showed no differences between the mutants and wild-type ICL, indicating that the conformations of the three enzymes were very similar. ICL C195A (Cys-195-->Ala) and C195S (Cys-195-->Ser) showed 8.4-fold and 3.6-fold increases in the Km for isocitrate, while their kcat. values showed 30- and 100-fold decreases respectively. The effect of pH on the kinetic properties of the wild-type and mutant ICLs was investigated. The results showed that the response of the mutant enzymes to pH was simpler than that of the wild-type. For the mutants, ionisation of a group with a pKa of approx. 7.8 affected the Km for isocitrate and kcat.. For the wild-type enzyme, these parameters were affected by the ionization of two or more groups, one of which is presumed to by cysteine-195. The results are consistent with the view that the previously identified group with a pKa of 7.1 whose ionization affects the reaction of ICL by iodoacetate is cysteine-195 itself.

Gamma-irradiation resistant mutants of Escherichia coli: modified enzyme synthesis repressible by arginine and uracil

1969 ◽  
Vol 15 (6) ◽  
pp. 549-554
Author(s):  
Haskel Robern ◽  
F. S. Thatcher
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Feedback Inhibition ◽  
Residual Activity ◽  
Wild Type ◽  
Aspartate Transcarbamylase ◽  
Γ Radiation ◽  
Dependent Activity ◽  
In The Wild

The levels of ammonia- or glutamine-dependent carbamyl phosphate synthetase (CPS), aspartate transcarbamylase (ATC), and ornithine transcarbamylase (OTC) were determined in wild-type Escherichia coli and in three mutants resistant to γ-irradiation designated as 1γ, 6γ, and 12γ. Enzyme assays were conducted on the cultures after growth in an arginine–uracil-free medium (AUF) or in AUF medium supplemented with arginine and uracil (C).The growth rate of the wild type and 1γ was the same in AUF or C medium but the levels of CPS, ATC, and OTC in the 1γ mutant ranged from two to four times as high as in the wild-type strain when grown in AUF medium. The 6γ mutant grew more slowly than the wild type and 1γ strains but its rate of growth was the same in all media. When this mutant was grown in AUF medium, the levels of CPS and OTC were as high as in the 1γ mutant but the level of ATC was about the same as in the wild-type strain. The 12γ mutant was the slowest growing and required both arginine and uracil. When we grew the culture in limiting amounts of uracil and excess arginine or vice versa, the ammonia-dependent activity of CPS was higher than in the wild-type strain, but glutamine-dependent CPS, OTC, and ATC were not synthesized by this mutant. Cultures grown in medium C showed only residual activity of all enzymes tested. These results suggest that (1) the 1γ, 6γ, and 12γ mutants have abnormally low pools of arginine and uracil; (2) the mechanism for feedback inhibition is more sensitive to ionizing radiation than the catalytic mechanism; (3) all damage caused by γ-radiation is at the gene level; (4) the locus controlling the synthesis of the ammonia-dependent activity of CPS is the least sensitive to γ-radiation, and is subject to cumulative repression by uracil and arginine.

scholarly journals Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain

1994 ◽  
Vol 300 (1) ◽  
pp. 111-115 ◽  
Author(s):  
M H Rider ◽  
K M Crepin ◽  
M De Cloedt ◽  
L Bertrand ◽  
L Hue
Keyword(s):  
Escherichia Coli ◽  
Skeletal Muscle ◽  
Fold Increase ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Wild Type ◽  
Fluorescence Measurements

Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to study its role in catalysis and/or substrate binding. The D130A mutant displayed a 30- to 140-fold decreased 2-kinase Vmax, depending on the pH, and a 30- and 60-fold increase in Km for MgATP and Fru-6-P respectively at pH 8.5 compared with the wild-type. Mutagenesis of Asp-130 to Ala had no effect on the 2-phosphatase activity, and fluorescence measurements indicated that the changes in kinetic properties of PFK-2 in the D130A mutant were not due to instability. The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+.

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