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J N Evans

Publications and source records attributed to J N Evans.

At least 19 recordsLinked to original sources
PubMed2001

New insights into the metal center of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

Metal binding properties for a series of metal-substituted forms of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, DAHPS(Tyr), have been followed by UV-vis and EPR spectroscopy. The results show that there are two metal species present at pH = 7.0 and these are coordinated in a distorted metal binding site with a mixed nitrogen and oxygen donor atom coordination set. There is no spectroscopic evidence for strong M-S interactions in this system at any pH. Metal saturation occurs at a substoichiometric ratio of 0.8-0.85 metal/monomer, and the binding trends mirror previously published enzyme activity profiles. There is a conformational change for CuDAHPS under basic conditions, and equivalent protein handling for apoDAHPS leads to apparent loss of metal binding ability. Addition of the substrate PEP does not alter the UV-vis spectra, but there are small changes in the EPR spectra of CuDAHPS(Tyr). Further addition of the substrate analogue A5P has no effect on either spectra. Taken together, these results serve to link previous studies on enzyme activity with the recently determined X-ray crystal structure for DAHPS(Phe) and represent the first detailed spectroscopic characterization of the metal binding properties of DAHPS(Tyr).

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed2001

Chemical shift mapping of shikimate-3-phosphate binding to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.

To facilitate evaluation of enzyme-ligand complexes in solution, we have isolated the 26-kDa N-terminal domain of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase for analysis by NMR spectroscopy. The isolated domain is capable of binding the substrate shikimate-3-phosphate (S3P), and this letter reports the localization of the S3P binding site using chemical shift mapping. Based on the NMR data, we propose that Ser23, Arg27, Ser197, and Tyr200 are directly involved in S3P binding. We also describe changes in the observed nuclear Overhauser effects (NOEs) that are consistent with a partial conformational change in the N-terminal domain upon S3P binding.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2001

Shikimate-3-phosphate binds to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the transfer of the enolpyruvyl moiety from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P). Mutagenesis and X-ray crystallography data suggest that the active site of the enzyme is in the cleft between its two globular domains; however, they have not defined which residues are responsible for substrate binding and catalysis. Here we attempt to establish the binding of the substrate S3P to the isolated N-terminal domain of EPSP synthase using a combination of NMR spectroscopy and isothermal titration calorimetry. Our experimental results indicate that there is a saturable and stable conformational change in the isolated N-terminal domain upon S3P binding and that the chemical environment of the S3P phosphorus when bound to the isolated domain is very similar to that of S3P bound to EPSP synthase. We also conclude that most of the free energy of S3P binding to EPSP synthase is contributed by the N-terminal domain.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2001

Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail.

Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft. Glyphosate appears to occupy the binding site of the second substrate of EPSP synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme.substrates complex. The elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2000

Determination of the pKa value of C115 in MurA (UDP-N-acetylglucosamine enolpyruvyltransferase) from Enterobacter cloacae.

The enzyme UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyltransferase (MurA) catalyzes the formation of enolpyruvyl-UDP-NAG, a precursor in peptidoglycan biosynthesis. The residue at position 115 in MurA has been proposed to act as a general acid in the enzymatic reaction. This is also the primary site of action of the antibiotic fosfomycin. In this paper, the pK(a) of Cys-115 has been determined to be 8.3, by titration of Enterobacter cloacae MurA with the alkylating agent iodoacetamide as a function of pH. Use of site-directed mutagenesis has established that only C115 is essential for catalysis, and the three other cysteine residues (C251, C354, and C381) are nonessential. Mass spectrometric analysis demonstrated that C115 is not alkylated at pH <7, but is alkylated significantly at pH >7. Measurement of the enzymatic inhibition by iodoacetamide as a function of pH showed maximum inhibition at pH >9, with a second-order rate constant of inhibition of 44 M(-)(1) s(-)(1) at pH 10. The presence of either one of the substrates did not influence the inactivation behavior, while the presence of both substrates resulted in a 5-fold reduction in the extent of alkylation. The covalent species that results from PEP bound to C115 of MurA exhibited 50-100-fold increased resistance against alkylation by iodoacetamide. These results imply that C115 is appreciably protonated at physiological pH and, therefore, is capable of acting as a proton donor in the enzyme-catalyzed reaction. However, it also implies that C115 is appreciably deprotonated at physiological pH also, whereupon the resultant thiolate nucleophile may play an important role in the formation of the covalent O-phosphothioketal species, whose role in catalysis is yet to be established.

Alkyl and Aryl Transferases↗
PubMed2000

Pediatric vocal fold paralysis: a long-term retrospective study.

OBJECTIVE: To review our experience of pediatric vocal fold paralysis (VFP), with particular emphasis on etiological factors, associated airway pathologic conditions, and treatment and prognostic outcomes. DESIGN: Retrospective case review of a cohort of patients presenting with VFP. SETTING: Tertiary referral center. PATIENTS: A consecutive sample of 102 patients presenting with VFP to Great Ormond Street Hospital for Children, London, England, over a 14-year period from 1980 to 1994. RESULTS: There was an almost equal distribution of unilateral (52% [n = 53]) and bilateral (48% [n = 49]) VFP. Iatrogenic causes (43% [n = 44]) formed the largest group, followed by idiopathic VFP (35% [n = 36]), neurological causes (16% [n = 16]), and finally birth trauma (5% [n = 5]). Associated upper airway pathologic conditions were noted in 66% (n = 23) of patients who underwent tracheotomy. Tracheotomy was necessary in only 57% (n = 28) of children with bilateral VFP. Prognosis was variable depending upon the cause, with neurological VFP having the highest rate of recovery (71% [5/7]) and iatrogenic VFP the lowest rate (46% [12/26]). CONCLUSION: Recovery after an interval of up to 11 years was seen in idiopathic bilateral VFP; this has significant implications when considering lateralization procedures in these patients.

Humans↗
PubMed1999

Purification, cloning, and heterologous expression of a catalytically efficient flavonol 3-O-galactosyltransferase expressed in the male gametophyte of Petunia hybrida.

Flavonols are plant-specific molecules that are required for pollen germination in maize and petunia. They exist in planta as both the aglycone and glycosyl conjugates. We identified a flavonol 3-O-galactosyltransferase (F3GalTase) that is expressed exclusively in the male gametophyte and controls the formation of a pollen-specific class of glycosylated flavonols. Thus an essential step to understanding flavonol-induced germination is the characterization of F3GalTase. Amino acid sequences of three peptide fragments of F3GalTase purified from petunia pollen were used to isolate a full-length cDNA clone. RNA gel blot analysis and enzyme assays confirmed that F3GalTase expression is restricted to pollen. Heterologous expression of the F3GalTase cDNA in Escherichia coli yielded active recombinant enzyme (rF3GalTase) which had the identical substrate specificity as the native enzyme. Unlike the relatively nonspecific substrate usage of flavonoid glycosyltransferases from sporophytic tissues, F3GalTase uses only UDP-galactose and flavonols to catalyze the formation of flavonol 3-O-galactosides. Kinetic analysis showed that the k(cat)/K(m) values of rF3GalTase, using kaempferol and quercetin as substrates, approaches that of a catalytically perfect enzyme. rF3GalTase catalyzes the reverse reaction, generation of flavonols from UDP and flavonol 3-O-galactosides, almost as efficiently as the forward reaction. The biochemical characteristics of F3GalTase are discussed in the context of a role in flavonol-induced pollen germination.

Amino Acid Sequence↗
PubMed1999

Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase.

The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported. Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions. These residues, while not essential for catalysis, are most likely important for substrate binding. All of the mutants are shown to be correctly folded by NMR spectroscopy.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1998

On the mechanism of 5-enolpyruvylshikimate-3-phosphate synthase.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP, a precursor for the aromatic amino acids. This paper examines a recent claim [Studelska, D. R., McDowell, L. M., Espe, M. P., Klug, C. A., and Schaefer, J. (1997) Biochemistry 36, 15555-15560] that the mechanism of EPSP synthase involves two covalent enzyme-intermediates, in complete contrast to a large body of literature that has already proven the involvement of a single noncovalent intermediate. The evidence in the paper of Studelska et al. is examined closely, and unequivocal proof is provided that those authors' NMR assignments to covalent structures are in error, and that in fact the species they observed were simply the product EPSP and a side-product EPSP ketal. Since those authors used rotational-echo double-resonance (REDOR) solid-state NMR to measure intermolecular and intramolecular distances in the proposed covalent intermediates, we have used REDOR to measure the same distances in enzyme-free and enzyme-bound preparations of purified EPSP, and enzyme-free preparations of purified EPSP ketal. The distance between the shikimate ring phosphorus atom and C8 in enzyme-free EPSP is 6.6 +/- 0.1 A, which lengthens to 7.4 +/- 0.1 A in the presence of the enzyme, and in enzyme-free EPSP ketal is 5.6 +/- 0.1 A. These are entirely consistent with those measured by Studelska et al., which were 7.5 +/- 0.5 A for a putative enzyme-enolpyruvyl species and 6.1 +/- 0.3 A for a putative enzyme-ketal species.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1998

Effects of sample preparation conditions on biomolecular solid-state NMR lineshapes.

Sample preparation conditions with the 46 kDa enzyme complex of 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, shikimate-3-phosphate (S3P) and glyphosate (GLP) have been examined in an attempt to reduce linewidths in solid-state NMR spectra. The linewidths of 31P resonances associated with enzyme bound S3P and GLP in the lyophilized ternary complex have been reduced to 150 +/- 12 Hz and 125 +/- 7 Hz respectively, by a variety of methods involving additives and freezing techniques.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1997

Comparative studies of phase-cycling schemes for multiple pi-pulse sequences.

Recently, a new phase cycling scheme was introduced by this laboratory for use in biological solid-state NMR experiments involving multiple pi-pulses with characteristics that suggested it may enhance the sensitivity of these kind of experiments (Y. Li and J. N. S. Evans, 1995, Chem. Phys. Lett. 241, 79 and Erratum, 1995, ibid. 246, 527; Y. Li and J. N. S. Evans, 1996, J. Magn. Reson. B 111, 296). The new sequence followed the supercycled concept proposed a decade ago for heteronuclear decoupling experiments. In this paper, more detailed experiments demonstrate that the claim of enhanced sensitivity was unfounded, and in fact the supercycle proposed differs little from the established XY-8 and XY-16 based supercycles.

Magnetic Resonance Spectroscopy↗
PubMed1997

Overexpression, purification, and characterization of tyrosine-sensitive 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase from Escherichia coli.

An overexpression system (pCR105) for DAHP synthase (Tyr) was constructed by cloning the aroF gene at the NdeI site of the pET-22b(+) translation vector, a plasmid expression vector that contains the T7 lac promoter. The enzyme was overexpressed, purified to > 90% purity (by SDS-polyacrylamide gel electrophoresis), and characterized. The protein was overexpressed at a level of 58% the total soluble cell protein (based on enzymatic activities). About 244 mg of pure enzyme was obtained from a 2-liter cell culture. So far, this is the highest yield reported for the isozyme DAHP synthase (Tyr). The enzyme showed a bell-shaped pH-activity profile, with a pH optimum at pH 7.0-7.5 and pK values of 6.10 and 8.92. Inhibition of the enzyme by tyrosine was specific with 50% inhibition observed at 9 microM tyrosine, pH 7.0. The specific activity of the enzyme increased with added metal and metal sensitivity increased with purity of the enzyme. Only substoichiometric amounts of Cu, Fe, and Zn were found in the pure enzyme and this result is consistent with sensitivity of the enzyme to added metal. Although treatment with EDTA inactivated the enzyme almost completely, the activity of the apoenzyme was restored to differing extents by a variety of metals including Mn2+, Cd2+, Co2+, Fe2+, Cu2+, Mg2+, and Zn2+. Both Fe2+ and Cu2+ only partially reactivated EDTA-treated enzyme. Reconstitution of EDTA-treated enzyme with either Cd2+ or Mn2+ gave 1 mol of metal per mole of enzyme monomer. KCN inactivated the enzyme to only 80% and added metals reactivated the CN-treated enzyme only to a small extent. These results confirm the importance of the metal in the enzymatic reaction.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1997

The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif.

The solution structure of a complex between a truncated form of HMG-I(Y), consisting of the second and third DNA binding domains (residues 51-90), and a DNA dodecamer containing the PRDII site of the interferon-beta promoter has been solved by multidimensional nuclear magnetic resonance spectroscopy. The stoichiometry of the complex is one molecule of HMG-I(Y) to two molecules of DNA. The structure reveals a new architectural minor groove binding motif which stabilizes B-DNA, thereby facilitating the binding of other transcription factors in the opposing major groove. The interactions involve a central Arg-Gly-Arg motif together with two other modules that participate in extensive hydrophobic and polar contracts. The absence of one of these modules in the third DNA binding domain accounts for its-100 fold reduced affinity relative to the second one.

Amino Acid Sequence↗
PubMed1997

Activation of ERK and JNK1 MAP kinases in cultured lung tissue.

The biochemical mechanisms responsible for lung cell growth and proliferation are not well defined during tissue injury. We previously showed stimulation of DNA synthesis in intact lung tissue cultured in vitro after exposure to elevated fractions of O2. By use of this in vitro model, the current study examined the enzymatic activities of the extracellular-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. ERK and JNK activities were transiently elevated in lung tissue cultured under serum-free conditions. Raf-1 kinase, the primary upstream activator of the ERK pathway, was also transiently activated, suggesting a receptor-mediated ERK activation. Phosphorylation of the guanine nucleotide exchange protein p170 son of sevenless further suggested a receptor-mediated activation of the ERK pathway. c-Fos and c-Jun expression, downstream targets of ERK and JNK, was dramatically increased in cultured tissue compared with uncultured tissue. After the initial transient activation, ERK and JNK could be reactivated with specific agonists, demonstrating that these signaling pathways were functional. These findings demonstrate activation of the ERK and JNK MAP kinase pathways in intact lung tissue and provide a model system to define signaling pathways involved in lung tissue remodeling injury.

Blotting, Western↗
PubMed1996

Hemodynamic and biochemical characteristics of the aorta in the WKY, SHR, WKHT, and WKHA rat strains.

This study was designed to characterize the hemodynamic and biochemical properties of the abdominal aorta in four genetically related inbred rat strains that express genetic hypertension and hyperactive behavior in varying combinations. These include (1) the spontaneously hypertensive rat (SHR), which is hypertensive, hyperactive, and hyperreactive to stress; (2) Wistar-Kyoto (WKY) rats, which express none of these traits; (3) WKHT rats, which are hypertensive but not hyperactive; and (4) WKHA rats, which are hyperactive and hyperreactive to stress, but normotensive. Together, these four strains allowed us to examine the structural and functional changes in the aorta in the hypertensive SHR, the most widely used animal model of genetic hypertension, while controlling for the variables of hyperactivity and hyperreactivity that are also expressed in the SHR. Four groups of animals of both sexes were studied: (1) WKY, n = 101, (2) WKHA, n = 33, (3) WKHT, n = 91, and (4) SHR, n = 28. Blood pressure (BP) was determined by tail plethysmography as well as direct intraarterial monitoring under anesthesia. Fixed specimens were prepared for histologic analysis and the wall thickness determined morphometrically. Quantification of soluble tissue protein, elastin, and collagen in the aortic tissue was determined by measuring leucine (leu), hydroxyproline (HP/leu), and desmosine (DES/leu). The hypertensive strains (SHR and WKHT) had significantly higher tail BP than the normotensive strains (WKY and WKHA)-WKY: 128.7 +/- 22.3; WKHA: 126.7 +/- 14.6; WKHT: 162.8 +/- 21.2; SHR: 164.2 +/- 36.1 (p < 0.0001). Additionally, intraaortic diastolic BP and mean BP were higher in SHR rats than in WKHT. Morphometric studies showed the media thickness in the SHR rats was significantly greater than in the WKY and WKHA rats and no different than in the WKHT rats. Significantly less of the aortic wall protein was present as elastin in the hypertensive rats (SHR and WKHT), as well as the hyperactive rats (WKHA), compared to rats that had neither trait (WKY). These studies provide new information regarding aortic structure and function in genetic hypertension using inbred strains to control for the hyperactivity/hyperreactivity traits that coexist with hypertension in the SHR. They reveal that hypertensive aortas have altered matrix proteins that cannot be explained simply on the basis of blood pressure alone.

Amino Acids↗
PubMed1996

The H385N mutant of 5-enolpyruvylshikimate-3-phosphate synthase: kinetics, fluorescence, and nuclear magnetic resonance studies.

The site-directed mutagenesis of histidine-385 of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is reported. The H385N mutant is compared with wild type by a number of methods. H385N was found to retain 6% activity. Kinetic parameters, including Km values for the natural substrates and Ki and Kd values for the inhibitor glyphosate, were found to be similar to wild type. Unlike wild-type enzyme, H385N EPSP synthase does not show accumulation of enzyme-bound product (EPSP) in the 13C NMR spectrum under equilibrium conditions. These results suggest that this H385N mutant is less catalytically competent than the previously studied H385Q mutant and that the N(epsilon)H of histidine may be involved in hydrogen bonding to another residue involved in complexing the substrates/ products.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1996

The hard-soft acid-base principle in enzymatic catalysis: dual reactivity of phosphoenolpyruvate.

In this paper, the chemical reactivity of C3 of phosphoenolpyruvate (PEP) has been analyzed in terms of density functional theory quantified through quantum chemistry calculations. PEP is involved in a number of important enzymatic reactions, in which its C3 atom behaves like a base. In three different enzymatic reactions analyzed here, C3 sometimes behaves like a soft base and sometimes behaves like a hard base in terms of the hard-soft acid-base principle. This dual nature of C3 of PEP was found to be related to the conformational change of the molecule. This leads to a testable hypothesis: that PEP adopts particular conformations in the enzyme-substrate complexes of different PEP-using enzymes, and that the enzymes control the reactivity through controlling the dihedral angle between the carboxylate and the C==C double bond of PEP.

3-Deoxy-7-Phosphoheptulonate Synthase↗