Effect of pH on hydride transfer by Escherichia coli dihydrofolate reductase

The kinetic isotope effect (KIE) on hydride transfer in the reaction catalysed by dihydrofolate reductase from Escherichia coli (EcDHFR) is known to be temperature dependent at pH 7, but essentially independent of temperature at elevated pH. Here, we show that the transition from the temperature-dependent regime to the temperature-independent regime occurs sharply between pH 7.5 and 8. The activation energy for hydride transfer is independent of pH. The mechanism leading to the change in behaviour of the KIEs is not clear, but probably involves a conformational change in the enzyme brought about by deprotonation of a key residue (or residues) at high pH. The KIE on hydride transfer at low pH suggests that the rate constant for the reaction is not limited by a conformational change to the enzyme under these conditions. The effect of pH on the temperature dependence of the rate constants and KIEs for hydride transfer catalysed by EcDHFR suggests that enzyme motions and conformational changes do not directly influence the chemistry, but that the reaction conditions affect the conformational ensemble of the enzyme prior to reaction and control the reaction though this route.     

Circularly permuted dihydrofolate reductase of E.coli has functional activity and a destabilized tertiary structure

Three circularly permuted variants of Escherichia coli dihydrofolatereductase genes were constructed. Linkers coding tri- and pentapeptideswere used to connect the natural 5'- and 3'-terminal ends. Onlyone variant of circularly permuted protein with tripeptide linkerand the cleavage of the peptide bond between 107 and 108 aminoacid residues was produced in a good yield. The expressed proteinwas insoluble in the cells, but at pH 8.0 and higher the isolatedprotein was soluble. Enzymatic assay and physical studies haveshown that permuted dihydrofolate reductase has a destabilizedtertiary structure. Only the addition of the natural substratesor Inhibitors lead to the protein with the native-like structureand functional activity.     

Effects of single-residue substitutions on negative cooperativity in ligand binding to dihydrofolate reductase

The effects of six amino acid substitutions in Lactobacillus casei dihydrofolate reductase, predominantly in the coenzyme binding site, on catalysis and on the negative cooperativity between NADPH and tetrahydrofolate binding have been determined. Replacement of Leu62, His64 or Leu54 by alanine has no effect on kcat, and produces only modest changes in negative cooperativity. Alanine substitution of His77, which interacts indirectly with the coenzyme adenine ring, leads to a doubling of the negative cooperativity and a consequent doubling of kcat. Replacement of Arg43, which interacts with the coenzyme 2'- phosphate, by alanine, or of Trp21, which interacts with the coenzyme nicotinamide ring, by histidine leads to a 20-100-fold decrease in negative cooperativity. In both mutants there is a decrease in kcat; isotope effects show that product release is largely rate-limiting in R43A, whereas in W21H hydride ion transfer is rate-limiting. 1H NMR has been used to obtain information on the extent of the structural changes produced by the substitutions. This varies from very local effects in H64A to very widespread effects in W21H. These changes are used as the basis for discussion of the mechanisms of the functional effects of the various substitutions. It is suggested that residues in helix C, beta- strand 3 and the beta3-beta4 loop may be involved in the transmission of effects between the coenzyme and substrate binding sites.      

Comparative stability of dihydrofolate reductase mutants in vitro and in vivo

Dihydrofolate reductase mutants with amino acid replacementsin the active center (Thr35 Asp mutant, Arg57 His mutant andthe mutant with triple replacement Thr35 Asp, Asn37 Ser, Arg57 His) were obtained by site-directed mutagenesis. The stabilizationeffect of trimethoprim and NADP·H on the protein tertiarystructure in vitro has been investigated. In the case of mutantswith a ‘weak’ tertiary structure (Thr35 Asp35 andthe triple mutant) the separate addition of ligands does notaffect their stability. The simultaneous addition of these ligandsto Thr35 Asp35 and the triple mutant leads to the large increasein their stability. A distinct correlation was found betweenthe in vitro studied stability of the mutant proteins to theurea- or heat-induced denaturation and the level of proteolyticdegradation of these mutants previously observed in vivo.     

Effects of the length of a glycine linker connecting the N-and C- termini of a circularly permuted dihydrofolate reductase

An important consideration in the construction of active and stablecircularly permuted proteins is the connective sequence that links thenative N- and C-termini. For this reason, various lengths of polyglycinelinkers (two, three, four, five and six glycines) were employed to connectthe original N- and C-termini of a circularly permuted construct ofEscherichia coli dihydrofolate reductase (DHFR) in which the new N-terminuswas Met16. Examination of the circular dichroism (CD) spectra,gel-filtration chromatography elution profiles, urea-induced unfoldingproperties and enzyme kinetics revealed that, among the linkers tested, alinker length of five glycines was the most favorable. The Vmax of thecircularly permuted variant with a five glycine linker (cpM16G5) was about20% that of wild-type DHFR, although far UV CD spectra, gel filtrationelution time, conformational stability and Km for the substratedihydrofolate and Kd for the coenzyme NADPH were comparable in the twoproteins. Another circularly permuted DHFR with a five glycine linker inwhich a new N-terminus was created at Leu24 (cpL24G5) was also constructedand assayed. The Vmax of cpL24G5 was almost the same as the wild-type,presumably due to the optimization of the glycine linker. The improvedactivity of the Leu24 permutant is probably due to the disruption of acatalytically important structure, the M20 loop, in the Met16 permutant.     

A cross-section of the fitness landscape of dihydrofolate reductase

In vitro molecular evolution is regarded as a hill-climbingon a fitness landscape in sequence space, where the ‘fitness’is a quantitative measure of a certain physicochemical propertyof a biopolymer. We analyzed a ‘cross-section’ ofthe enzymatic activity landscape of dihydrofolate reductase(DHFR) by using a method of analysis of a fitness landscape.We limited the sequence space of interest to the five-dimensionalsequence space, where the coordinate corresponds to the 1st,16th, 20th, 42nd and 92nd site in the DHFR sequence. Thirtysix mutants mapped into the limited sequence space were takenin the analysis. As a result, the cross-section is of the roughMt Fuji type based on the mutational additivity. The ratio ofthe mean slope to the roughness is 2.8 and the Z-score of theoriginal ratio against a distribution of random references is7.0, which indicates a large statistical significance. The existenceof such a cross-section was discussed in terms of the occurrenceprobability of sets of five sites distantly separated from eachother on the DHFR 3D structure. Our results support the effectivenessof the evolution strategy which exploits the accumulation ofadvantageous single point mutations in such a cross-section.     

The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E.coli

The role of domains in defining the equilibrium and kinetic folding properties of dihydrofolate reductase (DHFR) from Escherichia coli was probed by examining the thermodynamic and kinetic properties of a set of variants in which the chain connectivity in the discontinuous loop domain (DLD) and the adenosine-binding domain (ABD) was altered by permutation. To test the concept that chain cleavage can selectively destabilize the domain in which the N- and C-termini are resident, permutations were introduced at one position within the ABD, one within the DLD and one at a boundary between the domains. The results demonstrated that a continuous ABD is required for a stable thermal intermediate and a continuous DLD is required for a stable urea intermediate. The permutation at the domain interface had both a thermal and urea intermediate. Strikingly, the observable kinetic folding responses of all three permuted proteins were very similar to the wild-type protein. These results demonstrate a crucial role for stable domains in defining the energy surface for the equilibrium folding reaction of DHFR. If domain connectivity affects the kinetic mechanism, the effects must occur in the sub-millisecond time range.     

Effects of substitution of Thr63 by alanine on the structure and function of Lactobacillus casei dihydrofolate reductase

A mutant of Lactobacillus casei dihydrofolate reductase hasbeen constructed in which Thr63, a residue which interacts withthe 2'-phosphate group of the bound coenzyme, is replaced byalanine. This substitution does not affect k_cat, but producesan 800-fold increase in the K_m for NADPH, which reflects dissociationof NADPH from the enzyme-NADPH-tetrahydrofolate complex, anda 625-fold increase (corresponding to 3.8 kcal/mol) in the dissociationconstant for the enzyme-NADPH complex. The difference in magnitudeof these effects indicates a small effect of the substitutionon the negative cooperativity between NADPH and tetrahydrofolate.Stopped-flow studies of the kinetics of NADPH binding show thatthe weaker binding arises predominantly from a decrease in theassociation rate constant. NMR spectroscopy was used to comparethe structures of the mutant and wild-type enzymes in solution,in their complexes with methotrexate and with methotrexate andNADPH. This showed that only minimal structural changes resultfrom the mutation; a total of 47 residues were monitored fromtheir resolved ¹H resonances, and of these nine in the binarycomplex and six in the ternary differed in chemical shift betweenmutant and wild-type enzyme. These affected residues are confinedto the immediate vicinity of residue 63. There is a substantialdifference in the ³¹P chemical shift of the 2'-phosphate ofthe bound coenzyme, reflecting the loss of the interaction withthe side chain of Thr63. The only changes in nuclear Overhausereffects (NOEs) observed were decreases in the intensity of NOEsbetween protons of the adenine ring of the bound coenzyme andthe nearby residues Leu62 and Ile102, showing that the substitutionof Thr63 does cause a change in the position or orientationof the adenine ring in its binding site.     

Construction and characterization of a single polypeptide chain containing two enzymatically active dihydrofolate reductase domains

A single polypeptide chain containing two dihydrofolate reductase( D M ) sequences from Escherichia coli was constructed to determineif a repeat sequence fusion protein could be expressed in anactive form. The possibility that intersequence interactionscould play a significant role for this enzyme is suggested bythe results of Hall and Frieden (1989, Proc. NatlAcad. Sri.USA, 86, 3060-3064) who observed a substantial decrease in theyield of active enzyme when folded hi the presence of a largeC-terminal fragment. The fusion protein [DHFR(Cysl52Ghi)-De-DHFR(MetlGln)] was efficiently expressed in E.coli cells and hasan activity which is twice that of the wild-type enzyme in thestandard assay. The Michaelis constants of the fusion proteinfor the substrate, dihydrofolate and the cofactor, NADPH, areessentially unchanged from those of the wild-type protein. Theureainduced in vitro unfolding reaction of the fusion proteinat low concentrations was found to be fully reversible and followa three state model, suggesting that the two domains unfoldindependently. At higher protein concentrations the unfoldingtransition broadened and shifted to a higher urea concentration.Size-exclusion chromatography results are consistent with theformation of aggregates at the higher protein concentration,even in the absence of denaturant.     

Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminal region

We have mutated lysine 2 and arginine 7 in elongation factor Tu from Escherichia coli separately either to alanine or glutamic acid. The aim of the work was to reveal the possible interactions between the conserved N-terminal part of the molecule, which is rich in basic residues and aminoacyl-tRNA. The enzymatic characterization, comprising GDP and GTP temperature stability assays and measurement of nucleotide dissociation and association rate constants, GTPase activity and aminoacyl-tRNA binding, shows that position 2 is not involved in aminoacyl-tRNA binding, while position 7 is necessary to accomplish this activity. Furthermore, arginine 7 seems to play a role in regulating the binding of GTP. The three-dimensional structure of the ternary complex, EF-Tu:GTP:Phe-tRNAPhe, involving Thermus aquaticus EF- Tu and yeast Phe-tRNA(Phe), shows that Arg7 is in a position which permits salt bridge formation with Asp284, thus binding the N-terminus tightly to domain 2. We propose that this interaction is needed for aminoacyl-tRNA binding, and also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form.      

Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations

We have applied a global approach to enzyme active site exploration, where multiple mutations were introduced combinatorially at the active site of Type II R67 dihydrofolate reductase (R67 DHFR), creating numerous new active site environments within a constant framework. By this approach, we combinatorially modified all 16 principal amino acids that constitute the active site of this enzyme. This approach is fundamentally different from active site point mutation in that the native active site context is no longer accounted for. Among the 1536 combinatorially mutated active site variants of R67 DHFR we created, we selected and kinetically characterized three variants with highly altered active site compositions. We determined that they are of high fitness, as defined by a complex function consisting jointly of catalytic activity and resistance to trimethoprim. The k(cat) and K(M) values were similar to those for the native enzyme. The favourable Delta(DeltaG) values obtained (ranging from -0.72 to -1.08 kcal/mol) suggest that, despite their complex mutational pattern, no fundamental change in the catalytic mechanism has occurred. We illustrate that combinatorial active site mutagenesis can allow for the creation of compensatory mutations that could not be predicted and thus provides a route for more extensive exploration of functional sequence space than is allowed by point mutation.     

Production of the enzyme dihydrofolate reductase by methotrexate-resistant bacteria isolated from soil

Six bacterial cultures isolated from soil were capable of growing in the presence of methotrexate (MTX). Two strains, PFR-1 and 3, developed resistance to 500 μg cm^?3 MTX in the medium and produced elevated levels of the enzyme dihydrofolate reductase (EC 1.5.1.3): 2580 and 2702 U dm^?3 compared to the sensitive parent strains (28 and 35 U dm^?3). Isolate PFR-3 showed maximum enzyme production (4950 U dm^?3, specific activity 12.56 U mg^?1 in flasks and 5737 U dm^?3, specific activity 14.80 U mg^?1 in 5-dm³ fermenter) during exponential phase of growth (6 h) at 37°C and pH 7.0.     

Invariant amino acid replacement affects the dihydrofolate reductase function and its gene expression

Three different forms of dihydrofolate reductase (DHFR) fromEscherichia coli with amino acid replacements Thr35 Asp, Asn37 Ser and Arg57 His, and one form containing all three of thesechanges were obtained by oligonucleotide-directed mutagenesis.These amino acids are on the surface of the protein and twoof them (Thr35 and Arg57) are invariant for known sequencesof DHFR. Conversion of Asn37 Ser has no effect on the functionalactivity or the protein level in the cells. The Thr35 Asp replacementleads to a sharp decrease in the protein level, while the additionof a DHFR inhibitor, trimethoprim (Tmp), to the growth mediumincreases the level of DHFR in the ceus. There is a very smallquantity of DHFR with all three amino acid changes. The additionof Tmp to the growth medium also leads to an increase in themutant protein levels. The mutant with the Arg57 His replacementrenders the cells sensitive to Tmp, but the level of DHFR isthe same as for the wild-type protein. It is suggested thatthe invariant Thr35 is important for the stable conformationof DHFR whereas Arg57 is essential for protein activity. Variousstructural and functional aspects of these results are discussed.     

Internalization and translocation of a new chimaeric protein composed of Pseudomonas aeruginosaexotoxin A and mouse dihydrofolate reductase as a model system

The following references were inadvertantly omitted: Debinski.W, Siegall,C.B., Fitzgerald.D. and Pastan,I. (1991)Mol. Cell Biol. 11, 1751–1753 Prior.T.I., Fitzgerald.DJ. and Pastan,I. (1991) Cell, 64, 1017–1023 The author apologizes for these omissions.     

Expression and epitope tagging of the membrane anchor subunit (DmsC) of Escherichia coli dimethyl sulfoxide reductase

Escherichia coli dimethyl sulfoxide reductase is a heterotrimer comprising a catalytic subunit (DmsA), an electron transfer subunit (DmsB) and an integral membrane anchor subunit (DmsC). DmsC is not antigenic and the production of antibodies to this subunit has not been successful. We have tagged DmsC at the C-terminus with a dystrophin- specific amino acid sequence (dysp) to which antibodies are readily available. We were able to use this tagging technique to monitor expression and localization of DmsC in E. coli and non-muscle eukaryotic cells. Growth properties of wild-type E. coli, strain HB101, overexpressing DmsC:dysp suggest that the expression of DmsC is lethal to E. coli. The lethality could be overcome by utilizing an E. coli F0F1 ATPase mutant as the host. Growth conditions of culture density, duration of induction, temperature of incubation after induction and media conditions were investigated to optimize DmsC:dysp accumulation levels. In order to alleviate the problem arising from the toxicity of DmsC, expression in eukaryotic tissue culture was also explored. A plasmid expressing DmsC:dysp was transfected into COS-1 or McA-RH777 cells. The presence of expressed DmsC:dysp was confirmed using specific anti-dysp antibodies and immunofluorescence microscopy analysis revealed that the DmsC:dysp was localized to the endoplasmic reticulum. Expression of DmsC:dysp did not appear to be toxic to the eukaryotic cells. These data suggest methodologies to overcome lethality problems associated with the overexpression of integral membrane proteins like DmsC.      

多铜氧化酶在大肠杆菌中的分泌表达

生物胺是存在于发酵食品中的一类有机物,过量摄入会危害人体健康。多铜氧化酶中的某些酶具有降解多种生物胺的活性,在减控发酵食品中的氨(胺)类危害物方面具有良好的应用前景。研究多铜氧化酶的分泌表达,对酶的特性改造和工业化生产与应用具有重要意义。本研究通过在解淀粉芽孢杆菌来源的多铜氧化酶N端融合信号肽PhoA实现了多铜氧化酶在大肠杆菌中的分泌表达,胞外酶活为69.8 U/L。通过优化诱导条件和酶的分泌确定了多铜氧化酶最优发酵条件为诱导温度25℃、IPTG浓度0.05 mmol/L、诱导时菌体OD600=1.0、诱导6 h后添加150 mmol/L甘氨酸;发酵40 h时胞外多铜氧化酶酶活达到238.1 U/L,是优化前的3.4倍。     

1株牛源大肠埃希菌的分离及鉴定

目的分离及鉴定内蒙古自治区通辽市某牛场犊牛腹泻致病菌。方法无菌采集犊牛腹泻粪便,通过分离培养、染色镜检、致病性检测、药敏检测、生化鉴定、16S r RNA PCR鉴定、血清型鉴定及系统进化树的构建进行病原研究。结果致病菌的16S rRNA基因序列与大肠埃希菌的相似性在99%以上;生化鉴定该致病菌为大肠埃希菌的置信度为99%,在20种常用药品中仅对阿米卡星、庆大霉素、亚胺培南、美洛培南敏感;血清鉴定该大肠埃希菌菌株的血清型为O17。结论该致病菌鉴定为大肠埃希菌,该菌具有多重耐药性,有成为人畜共患病病原的潜在可能。该研究为通辽地区牛场流行的大肠埃希菌防控提供了参考。     

Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system

In an attempt to introduce a large peptide that is not normallytranslocated across membranes into the cytosol of eukaryoticcells, we created a new chimeric protein termed CEDH betweenPseudomonas aeruginosa exotoxin A (ETA) and a variant enzymeof Mus musculus dihydrofolate reductase (DHFR) with reducedaffinity for antifolates, ETA^1–413.DHFR^1–187.ETA^609–613.We have defined, genetically constructed and expressed the chimericprotein in Escherichia coli. We showed that the CEDH chimericprotein, purified to homogeneity on an immunoaffinity resin,confers a methotrexate-resistant phenotype to Chinese hamsterovary cells. Furthermore, the chimeric protein allowed the growthof dihydrofolate reductase-deficient Chinese hamster ovary cellsin the absence of hypoxanthine and thymidine. These resultsdemonstrated that the chimeric protein exhibited enzyme activityand possessed the tightly folded native structure, and thatthe DHFR protein can be selectively internalized and translocatedvia domains of exotoxin A. These data show that the ETA systemis an efficient system for the delivery of a variety of largepolypeptides into the cytosol without stress to the target cells,and extends the use of this delivery system to proteins thatare not normally translocated across membranes.     

A structure-based model for the halphilic adaptiation of dihydrofolate reductase from Halobacterium volcanii

‘Halophilic adaptation’ of proteins, i.e. the requirementfor high concentrations of monovalent ions for thermodynamicstability of proteins from halophilic organisms, is not fullyunderstood. In this work, an explanation for the halophilicbehavior of dihydrofolate reductase (h-DHFR) from Halobacteriumvolcanii is attempted, based on a model structure derived fromcomparative modeling to dihydrofolate reductase from Escherichiacoli. The model structure of h-DHFR shows an unique asymmetricalcharge distribution over the protein surface, with positivelycharged amino acids centered around the active site and negativecharges on the opposite side of the enzyme. This particularcharge distribution and the correlated molecular dipole arefunctionally relevant. The negative charges on the surface formclusters which are shielded at high salt concentrations; atlow salt, they repulse each other, thus destabilizing the protein.Results are in accordance with denaturation data and, thus,provide an explanation for the exceptional stability propertiesof h-DHFR.