Inhibition of staphylococcal alpha-toxin by covalent modification of an arginine residue

The effects of 1,2-cyclohexanedione and phenylglyoxal on staphylococcal alpha-toxin were studied. Modification of one arginine residue in alpha-toxin was sufficient to render the toxin nonhemolytic with no conformational change. Modified alpha-toxin did not protect cells from hemolysis by native alpha-toxin. An arginine residue is therefore at or near the binding site of alpha-toxin. Trypsin digestion of modified alpha-toxin generated a 20 kDa fragment which was isolated using a boric acid gel column. Upon regeneration, this 20 kDa fragment was not recognized by a population of antibodies which prevented alpha-toxin binding. The fragment was recognized by antibodies directed against post-binding events. However, the antibinding antibodies recognized the intact modified toxin. This leads us to conclude that antibinding determinants are not found directly in the binding site or are conformationally masked.     

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M-1 min-1 is obtained at pH 8.2 and 25 degrees C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.     

Functional role of a conserved arginine residue located on a mobile loop of alkanesulfonate monooxygenase

The structure of the flavin-dependent alkanesulfonate monooxygenase (SsuD) exists as a TIM-barrel structure with an insertion region located over the active site that contains a conserved arginine (Arg297) residue present in all SsuD homologues. Substitution of Arg297 with alanine (R297A SsuD) or lysine (R297K SsuD) was performed to determine the functional role of this conserved residue in SsuD catalysis. While the more conservative R297K SsuD possessed a lower k(cat)/K(m) value (0.04 ± 0.01 μM(-1) min(-1)) relative to wild-type (1.17 ± 0.22 μM(-1) min(-1)), there was no activity observed with the R297A SsuD variant. Each of the arginine variants had similar K(d) values for flavin binding as wild-type SsuD (0.32 ± 0.15 μM), but there was no measurable binding of octanesulfonate. The low levels of activity for the R297A and R297K SsuD variants correlated with the absence of any detectable C4a-(peroxy)flavin formation in stopped-flow kinetic studies. Single-turnover experiments were performed in the presence of SsuE to evaluate both the reductive and oxidative half-reaction. With wild-type SsuD a lag phase is observed following the reductive half-reaction by SsuE that represents flavin transfer or conformational changes associated with the binding of substrates. Evaluation of the Arg297 SsuD variants in the presence of SsuE showed no lag phase following reduction by SsuE, and the flavin was oxidized immediately following the reductive half-reaction. These results corresponded with a lack of detectable changes in the proteolytic susceptibility of R297A and R297K SsuD in the presence of reduced flavin and/or octanesulfonate, signifying the absence of a conformational change in these variants with the substitution of Arg297.     

NMR studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida have been reconstituted with 13C- and 15N-enriched FAD. The protein preparations were studied by 13C-NMR, 15N-NMR and 31P-NMR techniques in the oxidized and in the two-electron-reduced states. The chemical shift values are compared with those of free flavin in water or chloroform. It is shown that the pi electron distribution in oxidized free p-hydroxybenzoate hydroxylase is comparable to free flavin in water, and it is therefore suggested that the flavin ring is solvent accessible. Addition of substrate has a strong effect on several resonances, e.g. C2 and N5, which indicates that the flavin ring becomes shielded from solvent and also that a conformational change occurs involving the positive pole of an alpha-helix microdipole. In the reduced state, the flavin in p-hydroxybenzoate hydroxylase is bound in the anionic form, i.e. carrying a negative charge at N1. The flavin is bound in a more planar configuration than when free in solution. Upon binding of substrate the resonances of N1, C10a and N10 shift upfield. It is suggested that these upfield shifts are the result of a conformational change similar, but not identical, to the one observed in the oxidized state. The 13C chemical shifts of FAD bound to apo(salicylate hydroxylase) indicate that in the oxidized state the flavin ring is also fairly solvent accessible in the free enzyme. Addition of substrate has a strong effect on the hydrogen bond formed with O4 alpha. It is suggested that this is due to the exclusion of water from the active site by the binding of substrate. In the reduced state, the flavin is anionic. Addition of substrate forces the flavin ring to adopt a more planar configuration, i.e. a sp2-hybridized N5 atom and a slightly sp3-hybridized N10 atom. The NMR results are discussed in relation to the reaction catalyzed by the enzymes.     

Hydroxylation of o-halogenophenol and o-nitrophenol by salicylate hydroxylase

Salicylate hydroxylase [EC 1.14.13.1] from Pseudomonas putida catalyzed the formation of catechol from substrate analogues such as o-nitro-, o-amino-, o-iodo-, o-bromo-, and o-chloro-phenol by removing the ortho-substituted groups. They are converted into nitrite, ammonia, and halide ions, respectively. Kinetic parameters of these reactions were determined by spectrophotometric and polarographic methods. Hydroxylation of o-nitro- or o-iodophenol proceeds with the unusual stoichiometry of 2:1:1 for consumed NADH, O2-uptake, and catechol formed. Other ortho-substituted phenols examined also gave the same results. Like salicylate, these substrates perturb the absorption spectrum of salicylate hydroxylase in the visible region, indicating the formation of enzyme.substrate complexes. Titration experiments with ortho-substituted phenols gave the dissociation constants of the complexes. The complexes were quantitatively reduced with NADH or dithionite without detectable formation of the intermediates. The fact that one atom of 18O2 was incorporated into the produced catechol in hydroxylation of o-nitrophenol indicates that the reaction is of monooxygenase nature. It is concluded that salicylate hydroxylase cleaves the C-N and C-X bonds of ortho-substituted phenols.     

Affinity chromatography of Pseudomonas salicylate hydroxylase

In order to facilitate the purification of salicylate hydroxylase (salicylate 1-monooxygenase, EC 1.14.13.1) from Pseudomonas sp. RPP (ATCC 29351), an affinity chromatography procedure was developed employing immobilized salicylate as the affinity ligand. The immobilization was achieved by reacting p-aminosalicylate with the N-hydroxysuccinimide ester of Sepharose 4B-6-aminohexanoic acid. When the bacterial crude extract was chromatographed with this affinity column, salicylate hydroxylase was absorbed to the gel while the bulk of protein freely passed through. The absorbed enzyme was subsequently eluted from the affinity column by applying a 0–60 mm sodium salicylate gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzymatically most active fraction of the affinity effluent revealed salicylate hydroxylase was by far the most predominant protein but there were also small amounts of contaminating proteins. However, a virtually homogeneous enzyme preparation was obtained when the crude extract was first fractionated with a DE-52 anion-exchange column followed by the affinity step. The enzyme preparation obtained by this two-step procedure showed a specific activity of 14.9 units/mg and an A₄₅₀:A₃₇₂:A₂₈₀ of 1.01:1:10.23. Because most of the enzymes belonging to the class of external flavoprotein monooxygenase utilize salicylate analogs as substrates and share many other common properties, there is a strong possibility that the salicylate column may be useful for the purification of other member monooxygenases.     

Chemical modification of an arginine residue in the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by phenylglyoxal

Phenylglyoxal (PGO) was used as a reagent for chemical modification of the ATP-binding site of Ca2+-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum (SR-ATPase). When 1 mM PGO was reacted with SR-ATPase at 30°C at pH 8.5, PGO was bound to the ATPase molecule in two-to-one stoichiometry with concomitant loss of activity of the ATPase to form the phosphorylated intermediate (E-P). ATP and ADP prevented the binding of PGO and thereby protected the enzyme from inactivation. The SR membranes were labeled with [14C]PGO and then digested with pepsin to identify the attachment site of PGO. A 14C-labeled peptide (402lle-Arg*-Ser-Gly-Gln406) was purified to homogeneity by C18-reversed phase HPLC (Arg* denotes the binding site of [14C]PGO). These results indicate that Arg403 is located in the ATP binding site of the SR-ATPase.     

Functional implication of the sole arginine residue of ribosomal proteins L7/L12

Modification of the only arginine residue present in proteins L7/L12 fromEscherichia coli with phenylglyoxal, 2,3-butanedione and 1,2-cyclohexanedione is accompanied by functional alterations. The capacity of these proteins to promote polyphenylalanine synthesis and elongation factor G-dependent hydrolysis of GTP in L7/L12-depleted ribosomal cores is significantly decreased (more than 50%) on modification. Incubation of the butanedione- and cyclohexanedione-modified L7/L12 under regenerating conditions is accompanied by recovery of the original activity in polyphenylalanine synthesis. These results and the conservation of the arginine residue in eubacterial L7/L12-type proteins point to the functional implication of this arginine residue.     

Chemical modification of arginine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: a kinetic and fluorescence study

The flavoprotein p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was modified by several arginine-specific reagents. Modifications by 2,3-butanedione led to the loss of activity of the enzyme, but the binding of p-hydroxybenzoate and NADPH to the enzyme was little or not at all affected. However the formation of the enzyme-substrate complex of the modified enzyme was accompanied by an increase of the fluorescence of protein-bound FAD, in contrast to that of native enzyme which leads to quenching of the fluorescence. Enzyme modified by phenylglyoxal did not bind p-hydroxybenzoate nor NADPH. Quantification and protection experiments showed that two arginine residues are essential and a model is described which accounts for the results. Modification by 4-hydroxy-3-nitrophenylglyoxal reduced the affinity of the enzyme for the substrate and NADPH. The ligands offered no protection against inactivation. From this it is concluded that one arginine residue is essential at some stage of the catalysis. This residue is not associated with the substrate- or NADPH-binding site of the enzyme. Time-resolved fluorescence studies showed that the average fluorescence lifetime and the mobility of protein-bound FAD are affected by modification of the enzyme.     

Insertion of an arginine residue into the transmembrane segments corrects protein misfolding

Deletion of Phe-508 (DeltaF508) in cystic fibrosis transmembrane conductance regulator causes cystic fibrosis because of misfolding of the protein. P-glycoprotein (P-gp) containing the equivalent mutation (DeltaY490) is also misfolded but can be rescued with drug substrates. Whether rescue is due to direct binding of drug substrate to the transmembrane (TM) segments or to indirect effects on cellular protein folding pathways is still controversial. P-gp-drug substrate interactions likely involve hydrogen bonds. If the mechanism of drug rescue involves changes to TM packing then we should be able to identify suppressor mutations in the TM segments that can mimic the drug rescue effects. We predicted that an arginine residue in the TM segments predicted to line the drug-binding pocket of P-gp (I306(TM5) or F343(TM6)) might suppress DeltaY490 P-gp protein misfolding because it has the highest propensity to form hydrogen bonds. We show that R306(TM5) or R343(TM6) increased the relative amount of mature DeltaY490 P-gp by 6-fold. Most other changes to Ile-306 or Phe-343 did not enhance maturation of DeltaY490 P-gp. The I306R mutant also promoted maturation of misprocessed mutants that had mutations in the second nucleotide-binding domain (L1260A), the cytoplasmic loops (G251V, F804A), the linker region (P709A), or in TM segments (G300V, G722A). These results show that arginine residues in the TM domains can mimic the drug rescue effects and are effective suppressor mutations for processing mutations located throughout the molecule.     

Specific modification of the functional arginine residue in soybean trypsin inhibitor (Kunitz) by peptidylarginine deiminase

In order to elucidate the specificity of rabbit muscle peptidylarginine deiminase, which catalyzes the conversion of arginyl to citrullyl residues (Takahara, H., Oikawa, Y., and Sugawara, K. (1983) J. Biochem. (Tokyo) 94, 1945-1953), we examined the action of this enzyme on a variety of trypsin inhibitors by assay of residual trypsin-inhibiting activity. The enzyme rapidly abolished the activity of soybean trypsin inhibitor (Kunitz) (STI) in a process that was pseudo-first order with the rate dependent on enzyme concentration (second order rate constant = 5.0 X 10(4) M-1 S-1), whereas no detectable changes in activity were noted for other inhibitors tested. Inactivation of STI was due to the conversion of 1 arginine to a citrulline residue and was accompanied with a 0.2 unit decrease of the isoelectric point. There was no alteration of the molecular size and overall conformation of STI. Furthermore, analysis of modified STI indicated that arginine 63, known as the reactive site of STI, is the residue modified by peptidylarginine deiminase. Thus, peptidylarginine deiminase selectively catalyzes the deimination of the functional arginine residue of STI.     

The solid phase synthesis of peptides containing an arginine residue with an unprotected guanidine group

A new variant of the solid phase synthesis of arginine-containing peptides was proposed. The conditions for the attachment to the Wang polymer of {ie235-1}-Fmoc-arginine containing a protonated guanidine group were found. We demonstrated that this attachment is accompanied by neither racemization nor the attachment of the second Arg residue. Side reactions involving the guanidine group of arginine were studied, and methods for their prevention were proposed. The comparison of the carbodiimide method with a 1-hydroxybenzotriazole additive and a modified method with the use of Kastro’s reagent for the introduction of {ie235-2}-Fmoc-Arg residue with the unprotected guanidine group into the growing peptide chain demonstrated the advantages of the second method. Bradykinin and a peptide corresponding to the 584–591 sequence of the transmembrane gp41 from HIV-1 were synthesized by the method proposed here.     

Modification of an arginine residue of a base-nonspecific ribonuclease from Aspergillus saitoi.

1. A base-nonspecific ribonuclease from Aspergillus saitoi [RNase Ms, EC 3.1.4.23; molecular weight, 12,500] was modified with phenylglyoxal (PG) and 1,2-cyclohexanedione (CHD) in order to determine whether a single arginine residue was involved in the active site of the enzyme. 2. RNase Ms was inactivated by both PG and CHD with concomitant loss of one arginine residue. A competitive inhibitor of RNase Ms, 2',(3')-AMP, protected the enzyme from inactivation by PG. These findings strongly suggest that one arginine residue is involved in the active site of RNase Ms. 3. Difference CD spectra were measured at pH 5.5 for the binding of 2'-AMP and adenosine to native RNase Ms and the CHD- and PG-modified enzyme derivatives to determine the association constants. The arginine modification brought about a marked decrease in the binding affinity of 2'-AMP for the enzyme, but only a slight decrease for adenosine, suggesting that the arginine residue had interacted with the phosphate groups of the substrate.     

Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis

Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [7-14C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 x 10(4). On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.     

Intermediate and mechanism of hydroxylation of o-iodophenol by salicylate hydroxylase

Salicylate hydroxylase [EC 1.14.13.1] from Pseudomonas putida catalyzes the hydroxylation of salicylate, and also o-aminophenol, o-nitrophenol, and o-halogenophenols, to catechol. The reactions with these o-substituted phenols comprise oxygenative deamination, denitration, and dehalogenation, respectively. The reaction stoichiometry, as to NADH oxidized, oxygen consumed, and catechol formed, is 2 : 1 : 1, respectively. The mechanisms for the deiodination and oxygenation of o-iodophenol were investigated in detail by the use of I(+)-trapping reagents such as DL-methionine, 2-chlorodimedone, and L-tyrosine. The addition of the traps did not change the molar ratio of catechol formed to NADH oxidized, nor iodinated traps produced were in the incubation mixture. The results suggest that I+ was not produced on the deiodination in the hydroxylation of o-iodophenol. On the other hand, L-ascorbate, L-epinephrine, and phenylhydrazine increased the molar ratio. o-Phenylenediamine decreased it, being converted to phenazine. This suggests that o-benzoquinone is formed in the oxidation of o-iodophenol as a nascent product. The quinone was detected spectrophotometrically by means of the stopped-flow method. Kinetic analysis of the reactions revealed that o-benzoquinone is reduced nonenzymatically to catechol by a second molecule of NADH. A mechanism of elimination for the ortho-substituted groups of substrate phenols by the enzyme is proposed and discussed.     

Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene

Transgenic tobacco plants that express the bacterial nahG gene encoding salicylate hydroxylase have been shown to accumulate very little salicylic acid and to be defective in their ability to induce systemic acquired resistance (SAR). In recent experiments using transgenic NahG tobacco and Arabidopsis plants, we have also demonstrated that salicylic acid plays a central role in both disease susceptibility and genetic resistance. In this paper, we further characterize tobacco plants that express the salicylate hydroxylase enzyme. We show that tobacco mosaic virus (TMV) inoculation of NahG tobacco leaves induces the accumulation of the nahG mRNA in the pathogen infected leaves, presumably due to enhanced stabilization of the bacterial mRNA. SAR-associated genes are expressed in the TMV-infected leaves, but this is localized to the area surrounding necrotic lesions. Localized acquired resistance (LAR) is not induced in the TMV-inoculated NahG plants suggesting that LAR, like SAR, is dependent on SA accumulation. When SA is applied to nahG-expressing leave's SAR gene expression does not result. We have confirmed earlier reports that the salicylate hydroxylase enzyme has a narrow substrate specificity and we find that catechol, the breakdown product of salicylic acid, neither induces acquired resistance nor prevents the SA-dependent induction of the SAR genes.     

Studies of electron-transfer properties of salicylate hydroxylase from Pseudomonas cepacia and effects of salicylate and benzoate binding

The pH dependence of the redox behavior of salicylate hydroxylase from Pseudomonas cepacia as well as the effects of salicylate, benzoate, and chloride binding is described. At pH 7.6 in 0.02 M potassium phosphate buffer E1(0')(EFl ox/EFl.-) is -0.150 V and E2(0')(EFl.-/EFl red H-) is -0.040 V versus the standard hydrogen electrode (SHE). A maximum of 5% of FAD anion semiquinone is thermodynamically stabilized under these conditions. However, in coulometric and dithionite titrations more semiquinone is kinetically formed, indicating slow transfer of the second electron. The potential/pH dependence is consistent with a two-electron, one-proton transfer. Upon salicylate binding the midpoint potential is shifted 0.020 V negative from -0.094 to -0.114 V vs SHE at pH 7.6. A maximum of 7% of the neutral semiquinone is stabilized both in potentiometric and coulometric titrations. This small potential shift indicates that the substrate is bound nearly to the same extent to all three oxidation states of the enzyme. It is clear that the substrate binding does not make the reduction of the flavin thermodynamically more favorable. In contrast to salicylate, the potential shift caused by the effector, benzoate, is much more significant. (A maximum potential shift of -0.07 V is calculated.) Benzoate binds most tightly to the oxidized form and is least tightly bound to the two-electron-reduced form of the enzyme. For the reduction of the free enzyme the transfer of the second electron or the transfer of the proton is rate limiting, as is shown by the kinetic formation of the anionic semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous culture with complete cell recycle: cultivation of Pseudomonas cepacia ATCC 29351 on salicylate for production of salicylate hydroxylase

Summary Pseudomonas cepacia ATCC 29351 was cultivated on salicylate as sole carbon source in a system with complete cell recycling by means of a hollow fiber microfilter. Comparisons with batch cultures and continuous cultures are made with respect to cell densities, yield coefficients, specific enzyme activities and volumetric productivities. In batch cultures the toxicity of the salicylate limits the cell densities to below 1 g/l. In a recycling cultivation, a cell concentration of 15.1 g/l was obtained and the volumetric productivity of the cell mass was increased by a factor of 3. No serious effects on the cells were noted.     

Identification of an essential arginine residue in the beta subunit of the chloroplast ATPase

The ATPase activity of soluble chloroplast coupling factor (CF1) was irreversibly inactivated by phenylglyoxal, an arginine reagent. Under the conditions of inactivation, 2.48 mol of [14C]phenylglyoxal were incorporated per 400,000 g of enzyme when the ATPase was inactivated 50% by the reagent. Isolation of the component polypeptide subunits of the [14C]phenylglyoxal-modified enzyme revealed that the distribution of moles of labeled reagent/mol of subunit was the following: alpha, 0.37; beta, 0.40; gamma, 0.08; delta, none; epsilon, 0.03. CNBr treatment of the isolated alpha and beta subunits and fractionation of the peptides by gel electrophoresis revealed that the radioactivity bound to the alpha subunit was nonspecifically associated with several peptides, while a single peptide derived from the beta subunit contained the majority of the radioactivity associated with this subunit. After treating the isolated beta subunit with trypsin and Staphylococcus aureus protease, a major radioactive peptide was isolated with a sequence Arg-Ile-Thr-Ser-Ile-Lys. This sequence, when compared with the primary structure of the CF1 beta subunit as translated from the gene (Zurawski, G., Bottomley, W., and Whitfeld, P. R. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6260-6264) indicated that the arginine marked with the asterisk, the predominant residue modified by phenylglyoxal when the ATPase activity of CF1 is inactivated by the reagent, is Arg 312.