In vitro selection of an RNA sequence that interacts with high affinity with thymidylate synthase

Previous studies have shown that the repressive effect of thymidylate synthase (TS) mRNA translation is mediated by direct binding of TS itself to two cis-acting elements on its cognate mRNA. To identify the optimal RNA nucleotides that interact with TS, we in vitro synthesized a completely degenerate, linear RNA pool of 25 nt and employed in vitro selection to isolate high affinity RNA ligands that bind human TS protein. After 10 rounds of selection and amplification, a single RNA molecule was selected that bound TS protein with nearly 20-fold greater affinity than native, wild-type TS RNA sequences. Secondary structure analysis of this RNA sequence predicted it to possess a stem–loop structure. Deletion and/or modification of the UGU loop element within the RNA sequence decreased binding to TS by up to 1000-fold. In vivo transfection experiments revealed that the presence of the selected RNA sequence resulted in a significant increase in the expression of a heterologous luciferase reporter construct in human colon cancer H630 and TS-overexpressing HCT-C:His-TS+ cells, but not in HCT-C18 cells expressing a functionally inactive TS. In addition, the presence of this element in H630 cells leads to induced expression of TS protein. An immunoprecipitation method using RT–PCR confirmed a direct interaction between human TS protein and the selected RNA sequence in transfected human cancer H630 cells. This study identified a novel RNA sequence from a degenerate RNA library that specifically interacts with TS.     

Identification of cysteine-380 as the essential residue for the human N-acetyl-D-glucosamine 2-epimerase (renin binding protein).

Renin binding protein (RnBP) is a proteinous renin inhibitor firstly isolated from porcine kidney. Recently, the protein was identified as the enzyme, N-acetyl-D-glucosamine (GlcNAc) 2-epimerase. The GlcNAc 2-epimerase activity of recombinant human RnBP was specifically inhibited by SH-reagents such as N-ethylmaleimide, 5, 5'-dithiobis-2-nitrobenzoate, and iodoacetic acid, indicating that the most probable reactive site is a cysteine residue. To identify the active site residue(s), we have constructed ten cysteine residue mutants (C41S, C66S, C104S, C125S, C210S, C239S, C302S, C380S, C386S, and C390S) for human GlcNAc 2-epimerase and expressed them in Escherichia coli cells. The relative specific activities of C41S, C66S, C125S, C210S, C239S, C302S, C386S, and C390S are nearly the same to that of the wild-type enzyme. The specific activity of the C104S mutant is 26% of that of the wild-type enzyme. The expression of the C380S mutant in E. coli cells was detected on Western blotting, whereas GlcNAc 2-epimerase activity was not detected in the extract. These results indicate that Cys380 is essential for the enzymatic activity of human GlcNAc 2-epimerase.     

Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein from Escherichia coli: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes

The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme from Escherichia coli was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. The K_m values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.     

Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition

Previous studies have shown that human dihydrofolate reductase (DHFR) acts as an RNA-binding protein, in which it binds to its own mRNA and, in so doing, results in translational repression. In this study, we used RNA gel mobility shift and nitrocellulose filter-binding assays to further investigate the specificity of the interaction between human DHFR protein and human DHFR mRNA. Site-directed mutagenesis was used to identify the critical amino acid residues on DHFR protein required for RNA recognition. Human His-Tag DHFR protein specifically binds to human DHFR mRNA, while unrelated proteins including thymidylate synthase, p53 and glutathione-S-transferase were unable to form a ribonucleoprotein complex with DHFR mRNA. The Cys6 residue is essential for RNA recognition, as mutation at this amino acid with either an alanine (C6A) or serine (C6S) residue almost completely abrogated RNA-binding activity. Neither one of the cysteine mutant proteins was able to repress the in vitro translation of human DHFR mRNA. Mutations at amino acids Ile7, Arg28 and Phe34, significantly reduced RNA-binding activity. An RNA footprinting analysis identified three different RNA sequences, bound to DHFR protein, ranging in size from 16 to 45 nt, while a UV cross-linking analysis isolated an ~16 nt RNA sequence bound to DHFR. These studies begin to identify the critical amino acid residues on human DHFR that mediate RNA binding either through forming direct contact points with RNA or through maintaining the protein in an optimal structure that allows for the critical RNA-binding domain to be accessible.     

Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA

Thymidylate synthase (TS) functions as an RNA-binding protein by interacting with two different sequences on its own mRNA. One site is located in the 5′-upstream region of human TS mRNA while the second site is located within the protein coding region corresponding to nt 434–634. In this paper, a 70 nt RNA sequence, corresponding to nt 480–550, was identified that binds TS protein with an affinity similar to that of full-length TS mRNA and TS434–634 RNA. In vitro translation studies confirmed that this sequence is critical for the translational autoregulatory effects of TS. To document in vivo biological significance, TS sequences contained within this region were cloned onto the 5′-end of a luciferase reporter plasmid and transient transfection experiments were performed using H630 human colon cancer cells. In cells transfected with p644/TS434–634 or p644/TS480–550, luciferase activity was decreased 2.5-fold when compared to cells transfected with p644 plasmid alone. Luciferase mRNA levels were identical for each of these conditions as determined by RNase protection and RT–PCR analysis. Immunoprecipitation of TS ribonucleoprotein complexes revealed a direct interaction between TS protein and TS480–550 RNA in transfected H630 cells. Treatment with 5-fluorouridine resulted in a nearly 2-fold increase in luciferase activity only in cells transfected with p644/TS434–634 and p644/TS480–550. This study identifies a 70 nt TS response element in the protein coding region of TS mRNA with in vitro and in vivo translational regulatory activity.     

Characterization of an Escherichia coli mutant MutY with a cysteine to alanine mutation at the iron-sulfur cluster domain

Escherichia coli MutY is an adenine and a weak guanine DNA glycosylase involved in reducing mutagenic effects of 7,8-dihydro-8-oxoguanine (8-oxoG). The [4Fe-4S] cluster of MutY is ligated by four conserved cysteine residues and has been shown to be important in substrate recognition. Here, we show that the C199A mutant MutY is very insoluble and can be denatured and renatured to regain activity only if iron and sulfur are present in the renaturation steps. The solubility of C199A-MutY can be improved substantially as a fusion protein containing streptococcal protein G (GB1 domain) at its N-terminus. Here, we describe the first biochemical characterization of the purified GB1-C199A-MutY protein which contains a [3Fe-4S] cluster. The apparent dissociation constant (K(d)) values of GB1-C199A-MutY with both A/G and A/8-oxoG mismatches are slightly higher than that of the wild-type protein. The DNA glycosylase activity of GB1-C199A-MutY is comparable to that of the wild-type enzyme. Interestingly, the major difference between the C199A-MutY and wild-type proteins is their trapping activities (formation of Schiff base intermediates). The GB1-C199A-MutY mutant has a weaker trapping activity than the wild-type enzyme. Importantly, highly expressed GB1-C199A-MutY and untagged C199A-MutY can complement mutY mutants; however, GB1-C199A-MutY and untagged C199A-MutY cannot complement mutY mutants in vivo when both proteins are poorly expressed. Therefore, an intact [4Fe-4S] cluster domain is critical for MutY stability and activity.     

A computational study of RNA binding and specificity in the tandem zinc finger domain of TIS11d

TIS11d is a member of the CCCH-type family of tandem zinc finger (TZF) proteins; the TZF domain of TIS11d (residues 151–220) is sufficient to bind and destabilize its target mRNAs with high specificity. In this study, the TZF domain of TIS11d is simulated in an aqueous environment in both the free and RNA-bound states. Multiple nanosecond timescale molecular dynamics trajectories of TIS11d wild-type and E157R/E195K mutant with different RNA sequences were performed to investigate the molecular basis for RNA binding specificities of this TZF domain. A variety of measures of the protein structure, fluctuations, and dynamics were used to analyze the trajectories. The results of this study support the following conclusions: (1) the structure of the two fingers is maintained in the free state but a global reorientation occurs to yield a more compact structure; (2) mutation of the glutamate residues at positions 157 and 195 to arginine and lysine, respectively, affects the RNA recognition by this TIS11d mutant in agreement with the findings of Pagano et al. (J Biol Chem 2007; 282:8883–8894); and (3) we predict that the E157R/E195K mutant will present a more relaxed RNA binding specificity relative to wild-type TIS11d based on the more favorable nonsequence-specific Coulomb interaction of the two positively charged residues at positions 157 and 195 with the RNA backbone, which compensates for a partial loss of the stacking interaction of aromatic side chains with the RNA bases.     

Functional analysis at the Cys706 residue of the retinoblastoma protein.

A missense mutation at cysteine 706, resulting in a retinoblastoma (RB) protein defective in phosphorylation and oncoprotein binding, has been isolated from a human tumor cell line. Since this residue is conserved in murine RB and in the related p107 protein, we studied the activity of in vitro mutants flanking this position. These experiments demonstrated that the thiol atom at codon 706 does not possess intrinsic functional activity as small polar or nonpolar residues could substitute at either codons 706 or 707, while bulkier R-group changes in these positions interfered with in vitro oncoprotein binding or in vivo protein phosphorylation. A series of missense mutants in an adjacent leucine repeat domain also demonstrated a loss of oncoprotein binding that was proportional to the magnitude of amino acid substitutions. To determine whether the cysteine 706 --> phenylalanine RB mutant retained any protein binding activity, we examined its ability to precipitate MYC, which was recently identified as a potential RB-associated protein. These experiments demonstrated that the mutant RB product is capable of binding in vitro to c-myc and L-myc proteins with comparable affinity as wild-type RB. These findings raise questions about the functional role of the RB:MYC interactions and emphasize important differences in the binding patterns between MYC and the other RB-associated proteins.     

Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase II, a DExH box RNA helicase.

Vaccinia virus nucleoside triphosphate phosphohydrolase II (NPH-II), a 3'-to-5' RNA helicase, displays sequence similarity to members of the DExH family of nucleic acid-dependent nucleoside triphosphatases (NTPases). The contributions of the conserved GxGKT and DExH motifs to enzyme activity were assessed by alanine scanning mutagenesis. Histidine-tagged versions of NPH-II were expressed in vaccinia virus-infected BSC40 cells and purified by nickel affinity and conventional fractionation steps. Wild-type His-NPH-II was indistinguishable from native NPH-II with respect to RNA helicase, RNA binding, and nucleic acid-stimulated NTPase activities. The K-191-->A (K191A), D296A, and E297A mutant proteins bound RNA as well as wild-type His-NPH-II did, but they were severely defective in NTPase and helicase functions. The H299A mutant was active in RNA binding and NTP hydrolysis but was defective in duplex unwinding. Whereas the NTPase of wild-type NPH-II was stimulated > 10-fold by polynucleotide cofactors, the NTPase of the H299A mutant was nucleic acid independent. Because the specific NTPase activity of the H299A mutant in the absence of nucleic acid was near that of wild-type enzyme in the presence of DNA or RNA and because the Km for ATP was unaltered by the H299A substitution, we regard this mutation as a "gain-of-function" mutation and suggest that the histidine residue in the DExH box is required to couple the NTPase and helicase activities.     

Improved assay sensitivity of an engineered secreted Renilla luciferase.

We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2. The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity. In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues. Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity. Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase. This increase in light emission was due in part to enhanced stability of the mutant enzyme. This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression.     

Properties of bacteriophage T4 thymidylate synthase following mutagenic changes in the active site and folate binding region

Amino acid replacements have been introduced in specific sites of bacteriophage T4 thymidylate synthase (T4-TS) to assess the role that these changes have on enzyme activity. Each of the conserved amino acids in the active-site region of T4-TS was modified, and the effects that these changes had on the kinetic and physical properties of this enzyme were measured. The mutations introduced were Pro-155-Ala (P155A), Cys-156-Ser (C156S), and His-157-Val (H157V) with the resulting synthases possessing kcat's of 10.3, 0.008, and 2.70 s-1, respectively, relative to that of the wild-type enzyme of 11.8 s-1. Equilibrium dialysis was performed on the wild-type and mutant enzymes to determine the binding constants for 2'-deoxyuridylate and 5-fluoro-2'-deoxyuridylate, and while in most cases the extent of binding of these nucleotides to the mutant proteins was reduced when compared with wild-type TS, the number of binding sites involved remained about 1 or less for the binary complex and almost 2 for the ternary complex. Heat and urea stability studies revealed that the mutant with the highest enzyme activity, P155A, was the most unstable, while spectrofluorometric analyses revealed that the structures of P155A and H157V were perturbed relative to the C156S and wild-type TSs. These studies are in agreement with others implicating the phylogenetically conserved active-site cysteine as playing an essential mechanistic role in the catalytic process promoted by TS. The proximal amino acids on either side of this cysteine, although also highly conserved, do not appear to affect the catalytic mechanism directly, but may do so indirectly through their influence on the conformation at the active site as well as other regions of the enzyme. Amino acids replacements were introduced also into the folate and deoxynucleotide 5'-phosphate binding sites of the T4-phage TS to ascertain the potential role that these amino acids play in the catalytic process. These positions were selected on the basis of previous chemical modification and X-ray crystallographic studies on Lactobacillus casei TS. Amino acid residues 48 and 49, which are in the putative folate binding site, were converted from lysines to arginines; in the former case, the mutated enzyme had less than 7% of the wild-type activity while in the latter, the mutated enzyme still retained about 60% of its activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Substitution of Asp-210 in HAP1 (APE/Ref-1) eliminates endonuclease activity but stabilises substrate binding

HAP1, also known as APE/Ref-1, is the major apurinic/apyrimidinic (AP) endonuclease in human cells. Previous structural studies have suggested a possible role for the Asp-210 residue of HAP1 in the enzymatic function of this enzyme. Here, we demonstrate that substitution of Asp-210 by Asn or Ala eliminates the AP endonuclease activity of HAP1, while substitution by Glu reduces specific activity ~500-fold. Nevertheless, these mutant proteins still bind efficiently to oligonucleotides containing either AP sites or the chemically unrelated bulky p-benzoquinone (pBQ) derivatives of dC, dA and dG, all of which are substrates for HAP1. These results indicate that Asp-210 is required for catalysis, but not substrate recognition, consistent with enzyme kinetic data indicating that the HAP1–D210E protein has a 3000-fold reduced K_cat for AP site cleavage, but an unchanged K_m. Through analysis of the binding of Asp-210 substitution mutants to oligonucleotides containing either an AP site or a pBQ adduct, we conclude that the absence of Asp-210 allows the formation of a stable HAP1–substrate complex that exists only transiently during the catalytic cycle of wild-type HAP1 protein. We interpret these data in the context of the structure of the HAP1 active site and the recently determined co-crystal structure of HAP1 bound to DNA substrates.     

The NMR solution structures of the five constituent cold-shock domains (CSD) of the human UNR (upstream of N-ras) protein

Upon cold shock, the amounts of most proteins dramatically decrease from normal levels, but those of cold shock proteins (CSPs) and proteins containing cold-shock domains (CSDs) greatly increase. Although their biological function is still not completely clear, cold-shock proteins might control translation via RNA chaperoning. Many cold-shock proteins contain the motifs (Y/F)GFI and (V/F)(V/F)H, which are known as ribonucleoprotein (RNP)-1 and RNP-2 motifs implicated in RNA/DNA binding. We determined the solution NMR structures of all five constituent CSDs of the human UNR (upstream of N-ras) protein. The spatial arrangements of the sidechains in the RNP-1 and RNP-2 motifs are mostly conserved; however, the conformations of the following residues in the first CSD are different: F43 and H45 (the first phenylalanine residue and the histidine residue in the putative binding site RNP-2) and Y30 (the first residue in the putative binding site RNP-1). F43 and H45 affect each other, and H45 is further influenced by C46. The altered binding site of the first CSD, and its putatively enhanced intrinsic stability, may provide an explanation for the observation that the first CSD has 20-fold higher RNA-binding activity than the fifth CSD. It also lends support to the hypothesis that the UNR protein arose by repeated duplication of a protein that originally contained just one CSD, and that the proto-UNR protein acquired cysteine C46 by mutation during evolution.     

A single mutation within a Ca binding loop increases proteolytic activity,thermal stability,and surfactant stability

We improved the enzymatic properties of the oxidatively stable alkaline serine protease KP-43 through protein engineering to make it more suitable for use in laundry detergents. To enhance proteolytic activity, the gene encoding KP-43 was mutagenized by error-prone PCR. Screening identified a Tyr195Cys mutant enzyme that exhibited increased specific activity toward casein between pH 7 and 11. At pH 10, the mutant displayed 1.3-fold higher specific activity for casein compared to the wild-type enzyme, but the activity of the mutant was essentially unchanged toward several synthetic peptides. Furthermore, the Tyr195Cys mutation significantly increased thermal stability and surfactant stability of the enzyme under oxidizing conditions. Examination of the crystal structure of KP-43 revealed that Tyr195 is a solvent exposed residue that forms part of a flexible loop that binds a Ca^2 + ion. This residue lies 15–20 Å away from the residues comprising the catalytic triad of the enzyme. These results suggest that the substitution at position 195 does not alter the structure of the active center, but instead may affect a substrate–enzyme interaction. We propose that the Tyr195Cys mutation enhances the interaction with Ca^2 + and affects the packing of the Ca^2 + binding loop, consequently increasing protein stability. The simultaneously increased proteolytic activity, thermal stability, and surfactant stability of the Tyr195Cys mutant enzyme make the protein an ideal candidate for laundry detergent application.     

Cysteine 98 in CYP3A4 contributes to conformational integrity required for P450 interaction with CYP reductase

Previously human cytochrome P450 3A4 was efficiently and specifically photolabeled by the photoaffinity ligand lapachenole. One of the modification sites was identified as cysteine 98 in the B-C loop region of the protein [B. Wen, C.E. Doneanu, C.A. Gartner, A.G. Roberts, W.M. Atkins, S.D. Nelson, Biochemistry 44 (2005) 1833-1845]. Loss of CO binding capacity and subsequent decrease of catalytic activity were observed in the labeled CYP3A4, which suggested that aromatic substitution on residue 98 triggered a critical conformational change and subsequent loss of enzyme activity. To test this hypothesis, C98A, C98S, C98F, and C98W mutants were generated by site-directed mutagenesis and expressed functionally as oligohistidine-tagged proteins. Unlike the mono-adduction observed in the wild-type protein, simultaneous multiple adductions occurred when C98F and C98W were photolabeled under the same conditions as the wild-type enzyme, indicating a substantial conformational change in these two mutants compared with the wild-type protein. Kinetic analysis revealed that the C98W mutant had a drastic 16-fold decrease in catalytic efficiency (V(max)/K(m)) for 1'-OH midazolam formation, and about an 8-fold decrease in catalytic efficiency (V(max)/K(m)) for 4-OH midazolam formation, while the C98A and C98S mutants retained the same enzyme activity as the wild-type enzyme. Photolabeling of C98A and C98S with lapachenole resulted in monoadduction of only Cys-468, in contrast to the labeling of Cys-98 in wild-type CYP3A4, demonstrating the marked selectivity of this photoaffinity ligand for cysteine residues. The slight increases in the midazolam binding constants (K(s)) in these mutants suggested negligible perturbation of the heme environment. Further activity studies using different P450:reductase ratios suggested that the affinity of P450 to reductase was significantly decreased in the C98W mutant, but not in the C98A and C98S mutants. In addition, the C98W mutant exhibited a 41% decrease in the maximum electron flow rate between P450 and reductase as measured by reduced nicotinamide adenine dinucleotide phosphate consumption at a saturating reductase concentration. In conclusion, our data strongly suggest that cysteine 98 in the B-C loop region significantly contributes to conformational integrity and catalytic activity of CYP3A4, and that this residue or residues nearby might be involved in an interaction with P450 reductase.     

Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C³¹ and C¹⁰¹-X-X-C¹⁰⁴; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn²⁺ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C³¹ and C¹⁰¹-X-X-C¹⁰⁴, in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.     

Site-directed mutagenesis of rat liver NAD(P)H: quinone oxidoreductase: roles of lysine 76 and cysteine 179.

We previously reported the expression of a full-length cDNA complementary to a rat liver NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) mRNA in Escherichia coli (Q. Ma, R. Wang, C. S. Yang, and A. Y. H. Lu, 1990, Arch. Biochem. Biophys. 283, 311-317). Since cysteine residues have been suggested to be important for the catalysis of flavoproteins and a lysine residue at position 76 in NAD(P)H:quinone oxidoreductase has been proposed to be involved in electron transfer of the enzyme, we investigated the roles of lysine 76 and cysteine 179 of this enzyme in catalysis by site-directed mutagenesis. Mutant cDNA clones replacing lysine 76 with valine (K76V) and cysteine 179 with alanine (C179A) were generated by a procedure based on the polymerase chain reaction. The mutant enzymes were expressed in E. coli. The cytosolic activities of the K76V and C179A mutants were 50 and 25% of that of the wild type (DTD), due to lower levels of the mutant proteins as shown by immunoblot analysis. The mutant proteins were purified to apparent homogeneity. The purified K76V and C179A mutant enzymes maintained full activities of 2,6-dichlorophenolindophenol (DCIP) reduction compared with that of the wild type. The mutant enzymes exhibited kinetic parameters for DCIP, NADH, and NADPH similar to those of DTD except that, with K76V, the Km for NADPH was doubled. Both mutant proteins contained two molecules of FAD per enzyme molecule. Dicumarol inhibited K76V and C179A mutant activities to greater than 90% at a concentration of 10(-7) M. Heat stability studies showed that C179A was much more sensitive to inactivation at 37 degrees C than both the wild-type and K76V enzymes. It is concluded from this study that lysine 76 and cysteine 179 are not essential in catalysis and in the binding of FAD, DCIP, and dicumarol. However, lysine residue 76 appears to play a role in NADPH binding and cysteine residue 179 is important in maintaining the stability of the enzyme.     

Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues.

Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluorodeoxyuridylate (5-FdUMP), a tight binding covalent inhibitor of thymidylate synthase (TS). In order to create 5-FdUMP resistant enzymes to protect chemosensitive normal cells and further understand mechanisms of 5-FdUMP resistance, we have randomized four residues within the active site of TS. Our previous studies identified alterations in residues which produce active TS with enhanced resistance to 5-fluorouridine (5-FdUR). By remutagenizing a subset of the 13 previously targeted residues (A197, L198, C199 and V204), an unbiased random library can be created allowing for extensive testing of all possible amino acid substitutions at each of the sites. Using genetic complementation and selection in Escherichia coli, we identified the spectrum of substitutions that yield active TS as well as those that resulted in 5-FdUR resistant mutants of TS. The 5-FdUR resistant TS were found to share several structural features including hydrophobic substitutions at residue 197, retention of the wild-type leucine 198, the alteration C199L (present in 64% of the drug-resistant library), and polar alterations of valine 204. The catalytic activity of mutants with these features was approximately equal to that of the wild-type TS.     

Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA.

Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.