A second-site mutation at phenylalanine-137 that increases catalytic efficiency in the mutant aspartate-27----serine Escherichia coli dihydrofolate reductase

The adaptability of Escherichia coli dihydrofolate reductase (DHFR) is being explored by identifying second-site mutations that can partially suppress the deleterious effect associated with removal of the active-site proton donor aspartic acid-27. The Asp27----serine mutant DHFR (D27S) was previously characterized and the catalytic activity found to be greatly decreased at pH 7.0 [Howell et al. (1986) Science 231, 1123-1128]. Using resistance to trimethoprim (a DHFR inhibitor) in a genetic selection procedure, we have isolated a double-mutant DHFR gene containing Asp27----Ser and Phe137----Ser mutations (D27S+F137S). The presence of the F137S mutation increases kcat approximately 3-fold and decreases Km(DHF) approximately 2-fold over D27S DHFR values. The overall effect on kcat/Km(DHF) is a 7-fold increase. The D27S+F137S double-mutant DHFR is still 500-fold less active than wild-type DHFR at pH 7. Surprisingly, Phe137 is approximately 15 A from residue 27 in the active site and is part of a beta-bulge. We propose the F137S mutation likely causes its catalytic effect by slightly altering the conformation of D27S DHFR. This supposition is supported by the observation that the F137S mutation does not have the same kinetic effect when introduced into the wild-type and D27S DHFRs, by the altered distribution of two conformers of free enzyme [see Dunn et al. (1990)] and by a preliminary difference Fourier map comparing the D27S and D27S+F137S DHFR crystal structures.     

Role of aspartate 27 of dihydrofolate reductase from Escherichia coli in interconversion of active and inactive enzyme conformers and binding of NADPH

The apoenzyme of wild-type (WT) dihydrofolate reductase (DHRF) from Escherichia coli exists in two conformational states, Et and Ew, which differ in affinity for NADPH and in kinetic competence. Dissociation constants for the binary complex of NADPH with the two conformers differ by over 100-fold (KDt = 0.17 microM, KDw = 22 microM). Rate constants governing the interconversion of conformers are small (t1/2 for Ew----Et = 71 s), and since Ew is not catalytically competent, this conversion is accompanied by an increase in catalytic velocity. The equilibrium proportion of Et in the absence of ligands is 63%, but binding of NADPH greatly increases this proportion, and t1/2 for conversion of Ew.NADPH to Et.NADPH is 30 s. This conformational equilibrium has also been examined in mutant enzyme in which aspartate 27 is replaced by asparagine (D27N E. coli DHFR). Although ASp27 is an active site residue, it does not interact directly with bound NADPH, and in the mutant the rate constant for NADPH binding to Et is unchanged as are the dissociation constants for NADPH complexes with Et or Ew. However, for mutant apoenzyme, the proportion of Et is decreased to 18% in the absence of ligands so that the overall KD for NADPH is increased (0.15 microM for WT E. coli DHFR, 0.68 microM for D27N E. coli DHFR). The lower proportion of Et is due to a decreased rate for Ew----Et (t1/2 = 221 s) and an increased rate for Et----Ew (t1/2 = 50 s versus 120 s for WT E. coli DHFR).     

Dihydrofolate reductase from Lactobacillus casei. Stereochemistry of NADPH binding.

The NADPH molecule binds to dihydrofolate reductase in an extended conformation. Several of the individual dihedral angles, especially in the adenine mononucleotide portion of the coenzyme, differ from their minimum energy conformations. The ribose phosphate portions of the coenzyme are involved in numerous specific hydrogen-bonded and charge-charge interactions. The adenine ring resides in an apparently nonspecific hydrophobic cleft and the nicotinamide ring is bound within an intricately constructed cavity, one wall of which includes the pyrazine ring of bound methotrexate. Two rather extended loops (residues 10 to 24 and 117 to 135) connecting beta A to alpha B and beta F to beta G, respectively, move 2 to 3 A when NADPH binds to dihydrofolate reductase. No overall structural homology is evident between the dinucleotide binding domains of dihydrofolate reductase on the one hand and the four NAD+-dependent dehydrogenases of known structure on the other. However, binding does occur in both cases at the carboxyl edge of a region of parallel beta sheet flanked by a pair of alpha helices.     

Dihydrofolate reductase from Escherichia coli: the kinetic mechanism with NADPH and reduced acetylpyridine adenine dinucleotide phosphate as substrates

Kinetic studies on the reaction catalyzed by dihydrofolate reductase from Escherichia coli have been undertaken with the aim of characterizing further the kinetic mechanism of the reaction. For this purpose, the kinetic properties of substrates were determined by measurement of (a) initial velocities over a wide range of substrate concentrations and (b) the stickiness of substrates in ternary enzyme complexes. Stickiness is defined as the rate at which a substrate reacts to give products relative to the rate at which that substrate dissociates. Stickiness was determined by varying the viscosity of reaction mixtures and the concentration of one substrate in the presence of a saturating concentration of the other substrate. The results indicate that NADPH is sticky in the enzyme-NADPH-dihydrofolate complex, while dihydrofolate is much less sticky in this complex. At higher concentrations, NADPH functions as an activator through the formation of an enzyme-NADPH-tetrahydrofolate from which tetrahydrofolate is released more rapidly than from an enzyme-tetrahydrofolate complex. Higher concentrations of dihydrofolate also cause enzyme activation, and it appears that this effect is due to the ability of dihydrofolate to displace tetrahydrofolate from a binary enzyme complex through the formation of a transitory enzyme-tetrahydrofolate-dihydrofolate complex. As NADPH and dihydrofolate function as activators and as NADPH behaves as a sticky substrate, the kinetic mechanism of the dihydrofolate reductase reaction with the natural substrates is steady-state random. By contrast with NADPH, reduced 3-acetylpyridine adenine dinucleotide phosphate exhibits only slight stickiness and does not function as an activator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dihydrofolate reductase: the amino acid sequence of the enzyme from a methotrexate-resistant mutant of Escherichia coli.

The determination of the amino acid sequence of the enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) from a mutant of Escherichia coli B is described. The 159 residues were positioned by automatic Edman degradation of the whole protein, of the reduced and alkylated cyanogen bromide fragments, and of selected tryptic, chymotryptic, and thermolytic digestion products. An N-bromosuccinimide produced fragment of the largest cyanogen bromide peptide was also used in the sequence determination.     

Probing the functional role of phenylalanine-31 of Escherichia coli dihydrofolate reductase by site-directed mutagenesis

The role of Phe-31 of Escherichia coli dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Phe-31, a strictly conserved residue located in a hydrophobic pocket and interacting with the pteroyl moiety of dihydrofolate (H2F), was replaced by Tyr and Val. The kinetic behavior of the mutant enzymes in general is similar to that of the wild type. The rate-limiting step for both mutant enzymes is the release of tetrahydrofolate (H4F) from the E X NADPH X H4F ternary complex as determined for the wild type. The 2-fold increase in V for the two mutant enzymes arises from faster dissociation of H4F from the enzyme-product complex. The quantitative effect of these mutations is to decrease the rate of hydride transfer, although not to the extent that this step becomes partially rate limiting, but to accelerate the dissociation rates of tetrahydrofolate from product complexes so that the opposing effects are nearly compensating.     

Dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli: dihydrofolate monooxygenase activity

Dihydrofolate reductase from strain MB 1428 of Escherichia coli was shown to catalyze the oxidative cleavage of dihydrofolate at the C(9)N(10) bond. One of the products of the reaction was identified as 7,8-dihydropterin-6-carboxaldehyde through its proton magnetic resonance spectrum. The maximal enzymatic rate was 0.05 moles dihydrofolate cleaved per minute per mole enzyme at 25° and pH 7.2, and the K_M for dihydrofolate was 17.5 ± 2.5 μM. The enzymatic reaction was fully inhibitable with methotrexate. The mechanism of enzyme action was proposed to be an apparent “acidification” of dihydrofolate upon binding to the enzyme. Folate underwent an analogous oxidative cleavage by enzyme with a turnover number of 0.0014, which produced pterin-6-carboxaldehyde. Methotrexate was also slowly degraded by the enzyme.     

Role of aspartate 27 in the binding of methotrexate to dihydrofolate reductase from Escherichia coli

Dihydrofolate reductase from wild-type Escherichia coli (WT-ECDHFR) and from a mutant enzyme in which aspartate 27 is replaced by asparagine have been compared with respect to the binding of the inhibitor methotrexate (MTX). Although the Asp27----Asn substitution causes only small changes in the association rate constants (kon) for the formation of binary and ternary (with NADPH) complexes, the dissociation rate constants for these complexes (koff) are increased for the mutant enzyme by factors of about 5- and 100-fold, respectively, at pH 7.65. In binding experiments, the initial MTX binary and ternary complexes of the mutant enzyme were found to undergo relatively rapid isomerization (kobs approximately 17 and 145 s-1, respectively). Although such rapid isomerization of complexes of WT-ECDHFR could not be detected in binding experiments, evidence of a slow isomerization (k = 4 x 10(-3) s-1) of the ternary WT-ECDHFR.MTX.NADPH complex was obtained from progress of inhibition experiments. This slow isomerization increases binding of MTX to WT-ECDHFR only 2.4-fold (much less than previously estimated). From presently available data, we could not determine the contribution of the rapid isomerization of complexes to the binding of MTX to the mutant enzyme. The Asp27----Asn substitution increases the overall dissociation constant (KD) 9-fold for the binary complex and 85-fold for the ternary complex. When it is also taken into account that a proton ultimately derived from the solvent must be added to MTX bound to the WT enzyme, but not to MTX bound to the mutant enzyme, these increases in KD for the mutant enzyme correspond to decreases in binding energy for MTX of 3.9 and 5.2 kcal/mol at pH 7.65 for the binary and ternary complexes, respectively.     

Solution probing of metal ion binding by helix 27 from Escherichia coli 16S rRNA

Helix (H)27 from Escherichia coli 16S ribosomal (r)RNA is centrally located within the small (30S) ribosomal subunit, immediately adjacent to the decoding center. Bacterial 30S subunit crystal structures depicting Mg(2+) binding sites resolve two magnesium ions within the vicinity of H27: one in the major groove of the G886-U911 wobble pair, and one within the GCAA tetraloop. Binding of such metal cations is generally thought to be crucial for RNA folding and function. To ask how metal ion-RNA interactions in crystals compare with those in solution, we have characterized, using solution NMR spectroscopy, Tb(3+) footprinting and time-resolved fluorescence resonance energy transfer (tr-FRET), location, and modes of metal ion binding in an isolated H27. NMR and Tb(3+) footprinting data indicate that solution secondary structure and Mg(2+) binding are generally consistent with the ribosomal crystal structures. However, our analyses also suggest that H27 is dynamic in solution and that metal ions localize within the narrow major groove formed by the juxtaposition of the loop E motif with the tandem G894-U905 and G895-U904 wobble pairs. In addition, tr-FRET studies provide evidence that Mg(2+) uptake by the H27 construct results in a global lengthening of the helix. We propose that only a subset of H27-metal ion interactions has been captured in the crystal structures of the 30S ribosomal subunit, and that small-scale structural dynamics afforded by solution conditions may contribute to these differences. Our studies thus highlight an example for differences between RNA-metal ion interactions observed in solution and in crystals.     

Alteration of Escherichia coli topoisomerase IV conformation upon enzyme binding to positively supercoiled DNA

Escherichia coli topoisomerase IV (topo IV) is an essential enzyme that unlinks the daughter chromosomes for proper segregation at cell division. In vitro, topo IV readily distinguishes between the two possible chiralities of crossing segments in a DNA substrate. The enzyme relaxes positive supercoils and left-handed braids 20 times faster, and with greater processivity, than negative supercoils and right-handed braids. Here, we used chemical cross-linking of topo IV to demonstrate that enzyme bound to positively supercoiled DNA is in a different conformation from that bound to other forms of DNA. Using three different reagents, we observed novel cross-linked species of topo IV when positively supercoiled DNA was in the reaction. We show that the ParE subunits are in close enough proximity to be cross-linked only when the enzyme is bound to positively supercoiled DNA. We suggest that the altered conformation reflects efficient binding by topo IV of the two DNA segments that participate in the strand passage reaction.     

Dihydrofolate reductase from soybean seedlings: Characterization of the enzyme purified by affinity chromatography

Dihydrofolate reductase from soybean seedlings has been purified by agarose-formylaminopterin affinity chromatography. The enzyme is homogeneous as judged by disc gel electrophoresis and immunodiffusion. Analysis by both Sephadex G-200 column chromatography and Sephadex (superfine) G-200 thin-layer gel filtration gives a molecular weight of about 140,000 for the enzyme. Sodium dodecyl sulfate-gel electrophoresis reveals the presence of nonidentical subunits. The enzyme contains nine sulfhydryl groups and is inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and 5,5-dithiobis(2-nitrobenzoic acid). Folate analogs methotrexate, aminopterin, and formylaminopterin cause potent inhibition of the enzyme, with I₅₀ values (concentration required for 50% inhibition) of 0.25, 0.63, and 1.78 μm respectively. The turnover number of the enzyme is 57. K_m values for dihydrofolate and NADPH are 35 and 415 μm, respectively. Dihydrofolate, but not NADPH, affords protection against heat inactivation and the protection constant, K_p (concentration of dihydrofolate at which half the original activity is retained), is 81 μm.     

Dihydrofolate reductase: multiple conformations and alternative modes of substrate binding

The complex of Lactobacillus casei dihydrofolate reductase with the substrate folate and the coenzyme NADP+ has been shown to exist in solution as a mixture of three slowly interconverting conformations whose proportions are pH-dependent [Birdsall, B., Gronenborn, A. M., Hyde, E. I., Clore, G. M., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1982) Biochemistry 21, 5831]. The assignment of the resonances of all the aromatic protons of the ligand molecules in all three conformational states of the complex has now been completed by using a variety of NMR methods, particularly two-dimensional exchange experiments. The resonances of the nicotinamide protons of the coenzyme and the pteridine 7-proton of the folate have different chemical shifts in the three conformations, in some cases differing by more than 1 ppm. Comparison of the COSY spectra of the complex at low pH (conformation I) and high pH (conformations IIa and IIb) with that of the enzyme-methotrexate-NADP+ complex shows only slight differences in the conformation of the protein. The pattern of chemical shift changes in the ligand and the protein indicates that the structural differences are localized within the active site of the enzyme. Nuclear Overhauser effects (NOEs) are observed between the nicotinamide 5- and 6-protons and the methyl resonance of Thr 45 at both low and high pH, indicating that there is no major movement of the nicotinamide ring. By contrast, NOEs are observed between the pteridine 7-proton and the methyl protons of Leu 19 and Leu 27 in conformations I and IIa but not in conformation IIb.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ribonucleotide reductase from Escherichia coli: demonstration of a highly active form of the enzyme.

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The activity of the enzyme in crude extracts prepared from mechanically disrupted bacteria is very low. Enzyme activity is stimulated 5 to 10-fold by addition of an excess of either subunit. Concentrated extracts from cells lysed gently on Cellophane discs (Schaller et al.) contained 10 to 20-fold higher activity than extracts from mechanically disrupted cells. This activity was not further stimulated by either B1 or B2. The system is suitable for complementation tests for the analysis of temperature-sensitive mutants affecting the ribonucleotide reductase system. Concentrated high-speed supernatants from E. coli treated with lysozyme (Wickner et al.) also contained a high ribonucleotide reductase activity, which was stimulated slightly or not at all by addition of B1 and B2. This active form of the enzyme was unstable and could not be purified. The results suggest that the intracellular form of the enzyme consists of a tight complex of proteins B1 and B2, possibly stabilized by other intracellular structures.     

The role of exogenous iron in the activation of ribonucleotide reductase from Escherichia coli

 Protein R2, the small component of ribonucleotide reductase from Escherichia coli, contains a diferric center and a catalytically essential tyrosyl radical. In vitro, this radical can be produced in the protein from two inactive forms, metR2, containing an intact diiron center and lacking the tyrosyl radical, and apoR2, lacking both iron and the radical. While activation of apoR2 requires only a source of ferrous iron and exposure to O₂, activation of metR2 was achieved using a multienzymatic system consisting of an NAD(P)H:flavin oxidoreductase, superoxide dismutase and a poorly defined protein fraction, named fraction b (Fontecave M, Eliasson R, Reichard P (1987) J Biol Chem 262 : 12325–12331). In both reactions, reduced R2, containing a diferrous center, is a key intermediate which is subsequently converted to active R2 during reaction with O₂. By in vivo labeling of E. coli with radioactive ⁵⁹Fe, we show that fraction b contains iron. Depletion of the iron in fraction b inactivates it, and fraction b can be substituted for by ferric citrate solutions. Furthermore, aqueous Fe²⁺ in the presence of dithiothreitol is able to convert metR2 into reduced R2. Therefore we propose that the function of fraction b is to provide, in association with the flavin reductase, ferrous iron for reduction of the endogenous diiron center. Since fraction b is not a single well-defined protein, it remains to be shown whether, in vivo, that function resides in a specific protein. Exogenous iron can thus participate in activation of both apoR2 and metR2, but it is incorporated into R2 only in the former case. A unifying mechanism is proposed. Received: 13 November 1996 / Accepted: 3 April 1997