Development of a fluorescently labeled thermostable DHFR for studying conformational changes associated with inhibitor binding

A fluorescently-labeled, conformationally-sensitive Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR) (C73A/S131C_MDCC DHFR) was developed and used to investigate kinetics and protein conformational motions associated with methotrexate (MTX) binding. This construct bears a covalently-attached fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) attached at a distal cysteine, introduced by mutagenesis. The probe is sensitive to the local molecular environment, reporting on changes in the protein structure associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) also showed changes upon MTX association. Stopped-flow analysis of all data can be understood by invoking the presence of two native state DHFR conformers that bind to MTX at different rates (20.2 and 0.067 μM⁻¹ s⁻¹), similar to previously published findings for Escherichia coli DHFR. Probe fluorescence of C73A/S131C_MDCC DHFR predominantly reports on MTX binding to one of the conformers while intrinsic tryptophan fluorescence of C73A/S131C DHFR reports on binding to the other conformer. This study demonstrates the use of an extrinsic fluorophore attached to a distal region to investigate ligand binding interactions that are not experimentally accessible via intrinsic tryptophan fluorescence alone. The thermostability of C73A/S131C_MDCC DHFR provides an important new tool with applications for investigating the temperature dependence of DHFR conformational changes associated with binding and catalysis.     

Molecular characterization of the 5S ribosomal gene of the <Emphasis Type="Italic">Bradysia hygida</Emphasis>(Diptera:Sciaridae)

The rRNA genes are amongst the most extensively studied eukaryotic genes. They contain both highly conserved and rapidly evolving regions. The aim of this work was to clone and to sequence the Bradysia hygida 5S rDNA gene. A positive clone was sequenced and its 346 bp sequence was analyzed against the GenBank database. Sequence analysis revealed that the B. hygida 5S (Bh5S) rDNA gene is 120 bp long and is 87% identical to the aphid Acyrthosiphon magnoliae 5S rDNA gene. The Bh5S rDNA gene presents two unusual features: a GG pair at the 5' end of the gene sequence and the localization of the polyT signal immediately after the 3' end of the gene. In situ5S hybridization experiments revealed that the Bh5S rDNA gene is localized in the autosomal A chromosome     

Multicomplex-based pharmacophore modeling in conjunction with multi-target docking and molecular dynamics simulations for the identification of PfDHFR inhibitors

Abstract

Plasmodium falciparum dihydrofolate reductase enzyme (PfDHFR) is counted as one of the attractive and validated antimalarial drug targets. However, the point mutations in the active site of wild-type PfDHFR have developed resistance against the well-known antifolates. Therefore, there is a dire need for the development of inhibitors that can inhibit both wild-type and mutant-type DHFR enzyme. In the present contribution, we have constructed the common feature pharmacophore models from the available PfDHFR. A representative hypothesis was prioritized and then employed for the screening of natural product library to search for the molecules with complementary features responsible for the inhibition. The screened candidates were processed via drug-likeness filters and molecular docking studies. The docking was carried out on the wild-type PfDHFR (3QGT); double-mutant PfDHFR (3UM5 and 1J3J) and quadruple-mutant PfDHFR (1J3K) enzymes. A total of eight common hits were obtained from the docking calculations that could be the potential inhibitors for both wild and mutant type DHFR enzymes. Eventually, the stability of these candidates with the selected proteins was evaluated via molecular dynamics simulations. Except for SPECS14, all the prioritized candidates were found to be stable throughout the simulation run. Overall, the strategy employed in the present work resulted in the retrieval of seven candidates that may show inhibitory activity against PfDHFR and could be further exploited as a scaffold to develop novel antimalarials.

Communicated by Ramaswamy H. Sarma     

Vestigial mutants of Drosophila melanogaster live better in the presence of aminopterin: increased level of dihydrofolate reductase in a mutant

Summary Vestigial (vg) mutants of Drosophila melanogaster are characterized by atrophied wings. In this paper we show that: (1) aminopterin an inhibitor of dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase induce nicks in the wings of wild-type flies and phenocopies of the vg mutant phenotype when vg/+ and vg ^B/+ flies are reared on these substances (vg^B is a deficiency of the vg locus). Only thymidine and thymidylate can rescue the flies from the effect of aminopterin. We propose that the vg phenotype is due to a decrease in the dTMP pool in the wings. (2) Mutant vg strains yield more offspring on medium containing aminopterin than on normal medium. The resistance of vg larvae to the inhibitor seems specific to the gene. This is the first case of aminopterin resistance in living eucaryotes. In contrast sensitivity of the vg larvae to FUdR is observed. (3) An increase in the activity and amount of DHFR is observed in mutant strains as compared with the wild-type flies.Our data suggest that the vg ⁺ gene is a regulatory gene acting on the DHFR gene or a structural gene involved in the same metabolic pathway.     

Virtual screening,docking, and dynamics of potential new inhibitors of dihydrofolate reductase from Yersinia pestis

In the present work, we propose to design drugs that target the enzyme dihydrofolate redutase (DHFR) as a means of a novel drug therapy against plague. Potential inhibitors of DHFR from Yersinia pestis (YpDHFR) were selected by virtual screening and subjected to docking, molecular dynamics (MD) simulations, and Poisson–Boltzmann surface area method, in order to evaluate their interactions in the active sites of YpDHFR and human DHFR (HssDHFR). The results suggested selectivity for three compounds that were further used to propose the structures of six new potential selective inhibitors for YpDHFR.     

The influence of glycerol on the binding of methotrexate to Mycobacterium tuberculosis dihydrofolate reductase: a molecular modelling study

The enzyme, dihydrofolate reductase (DHFR), from Mycobacterium tuberculosis (mt-DHFR) is believed to be a potential drug target for the treatment of tuberculosis. The co-crystal structure of mt-DHFR bound with glycerol (GOL), NAPDH and methotrexate (MTX) reveals a GOL binding site on the enzyme. This GOL binding site could be very important for the design of novel, selective mt-DHFR inhibitors, because this binding site is absent on human DHFR (h-DHFR). We have performed molecular dynamic simulations and free energy calculations to evaluate the binding affinity of GOL and its free energy contribution to the binding of MTX to mt-DHFR. The results showed that GOL does not bind tightly to mt-DHFR. Although GOL itself contributed free energy on MTX binding to mt-DHFR, GOL also increased the flexibilities of MTX, so that MTX cannot maintain strong electronic interactions with ARG32 and ARG60, which caused the total binding free energy to decrease. These data suggest that GOL binding is weak and it could be expelled from the binding site, to allow inhibitors containing appropriate side chains to bind. This observation can be used to inform future drug design studies, especially those aimed at improving drug selectivity against h-DHFR.     

Selection for methotrexate resistance in mammalian cells bearing a <Emphasis Type="Italic">Drosophila</Emphasis> dihydrofolate reductase transgene

Antifolates, such as methotrexate (MTX), are the treatment of choice for numerous cancers. MTX inhibits dihydrofolate reductase (DHFR), which is essential for cell growth and proliferation. Mammalian cells can acquire resistance to antifolate treatment through a variety of mechanisms but decreased antifolate titers due to changes in drug efflux or influx, or alternatively, the amplification of the DHFR gene are the most commonly acquired resistance mechanisms. In Drosophila, however, a resistant phenotype has only been observed to occur by mutation resulting in a MTX-resistant DHFR. It is unclear if differences in gene structure and/or genome organization between Drosophila and mammals contribute to the observed differences in acquired drug resistance. To investigate if gene structure is involved, Drosophila Dhfr cDNA was transfected into a line of CHO cells that do not express endogenous DHFR. These transgenic cells, together with wild-type CHO cells, were selected for 19 months for resistance to increasing concentrations of MTX, from 50- to 200-fold over the initial concentration. Since Drosophila Dhfr appears to have been amplified several fold in the selected transgenic mammalian cells, a difference in genome organization may contribute to the mechanism of MTX resistance.     

Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h) DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (K_i 49 nM) compared to hDHFR (K_i 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show K_i values of 593 and 617 nM, respectively; these K_i values are well above both the K_i for pjDHFR and are similar to pcDHFR (Q35K/N64F and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes.     

A maize Ds transposable element containing a dihydrofolate reductase gene transposes in Nicotiana tabacum and Arabidopsis thaliana

Summary A mouse dihydrofolate reductase gene (DHFR), encoding an enzyme conferring methotrexate (MTX) resistance, under the control of the cauliflower mosaic virus (CaMV) 35 S promoter, was inserted within a maize nonautonomous Ds transposable element. The presence of at least one element (Ds-DHFR) can easily be monitored using methotrexate selection in plants. This chimeric element is able to transpose at a frequency similar to its unmodified progenitor in transgenic tobacco callus containing an autonomous Ac element. The orientation of the selectable marker cassette in the Ds element does not affect relative excision frequencies. Approximately two-thirds of these elements can be detected after excision while the remaining one-third cannot. The Ds-DHFR element is useful in elucidating the mechanism by which Ac/Ds transposition occurs, and allows for a rapid identification of mutants in which methotrexate resistance cosegregates with a mutant phenotype.     

Analysis of Coxiela burnetti dihydrofolate reductase via in silico docking with inhibitors and molecular dynamics simulation

Coxiella burnetii is a gram-negative bacterium able to infect several eukaryotic cells, mainly monocytes and macrophages. It is found widely in nature with ticks, birds, and mammals as major hosts. C. burnetii is also the biological warfare agent that causes Q fever, a disease that has no vaccine or proven chemotherapy available. Considering the current geopolitical context, this fact reinforces the need for discovering new treatments and molecular targets for drug design against C. burnetii. Among the main molecular targets against bacterial diseases reported, the enzyme dihydrofolate reductase (DHFR) has been investigated for several infectious diseases. In the present work, we applied molecular modeling techniques to evaluate the interactions of known DHFR inhibitors in the active sites of human and C. burnetii DHFR (HssDHFR and CbDHFR) in order to investigate their potential as selective inhibitors of CbDHFR. Results showed that most of the ligands studied compete for the binding site of the substrate more effectively than the reference drug trimethoprim. Also the most promising compounds were proposed as leads for the drug design of potential CbDHFR inhibitors.     

Molecular Cloning,Nucleotide Sequence,and Expression of the Structural Gene for Alkaline Serine Protease from Alkaliphilic Bacillus sp. 221

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus suhtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6—61.70/0). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.     

Cytological peculiarities of some extremely halophilic soil archaeobacteria

Ultrastructure and morphogenesis of extremely halophilic neutrophilic (Halobacteriam distributum, Halococcus turkmenicus) and alkaliphilic (Natronobacterium pharaonis, Natronococcus occultus) archaeobacteria were studied. The H. distributum culture was rather polymorphous and produced cells of four types. Due to the irregular cell fission in different planes packages of various numbers of cells surrounded by a common capsule were formed. Resting forms (halocysts) with multilayer covers were present in the population. The N. pharaonis culture consisted of rod-like cells and cyst-like forms. Besides, under conditions of carbon limitation, multicellular aggregated forms were found in the culture. Encapsulated single cells and aggregated forms with a common capsule were observed in H. turkmenicus and N. occultus cultures.     

Bone morphogenetic protein (BMP)1-3 enhances bone repair

Small molecules that exhibit biological activity have contributed to the understanding of the molecular mechanisms of various biological phenomena. 5-Bromodeoxyuridine (BrdU) is a thymidine analogue that modulates various biological phenomena such as cellular differentiation and cellular senescence in cultured mammalian cells. Although BrdU is thought to function through changing chromatin structure and gene expression, its precise molecular mechanisms are not understood. To study the molecular mechanism for the action of BrdU, we have employed the yeast Saccharomycescerevisiae as a model system, and screened multi-copy suppressor genes that confer resistance to BrdU. Our genetic screen has revealed that expression of the N-terminal short fragment of TUP1, and also disruption of HDA1 or HOS1, histone deacetylases that interact with TUP1, conferred resistance to BrdU. These results suggest the implication of the chromatin proteins in the function of BrdU, and would provide novel clues to answer the old question of how BrdU modulates various biological phenomena.     

Studies on the respiratory system in alkaliphilic Bacillus; a proposed new respiratory mechanism

Respiratory electron transfer systems in two alkaliphilic Bacillus species, YN-1 and YN-2000, were investigated. In the cyanide-sensitive pathway of the obligate alkaliphilic Bacillus YN-1, the terminal enzyme was a caa ₃-type cytochrome c oxidase constituting up to just 10% of the total oxygen-reducing activity, while 90% of the respiratory activity was due to cyanide-insensitive, nonproteinaceous material with a molecular weight of 662. These results were consistent with the cyanide-tolerant growth of the bacterium. The molecular and catalytic properties of the nonproteinaceous material were not identical with those of menaquinones extracted from the bacterium. Furthermore, the nonproteinaceous material was also found in the facultative alkaliphilic Bacillus YN-2000, when that bacterium was cultivated in alkaline conditions. A new respiratory oxygen-reducing mechanism comprising a nonproteinaceous component and a catalase is proposed for these alkaliphilic Bacillus species. Received: October 31, 1997 / Accepted: December 17, 1997     

Analysis of variants affecting the catalase developmental program in maize scutellum

Summary The catalase of maize scutella is coded for by two loci, Cat1 and Cat2, which are differentially expressed in this tissue during early seedling growth. Two variant lines have been previously identified in which the developmental program for the expression of the Cat2 structural gene in the scutellum has been altered. Line R6–67 exhibits higher than normal levels of CAT-2 catalase in this tissue after four days of postgerminative growth. This phenotype is controlled by a temporal regulatory gene designated Car1. Line A16 exhibits a CAT-2 null phenotype. Further analysis of Car1 verifies the initial indication that it is trans-acting and exhibits strict tissue (scutellum) specificity. A screen of other available inbred lines uncovered eight additional catalase high-activity lines. All eight lines exhibit significantly higher than normal levels of CAT-2 protein. Two of these lines have been shown to be regulated by Car1 as in R6–67. Another line (A338) uncovered during the screen exhibits a null phenotype for CAT-2 protein and resembles A16. Catalase activity levels are low in the scutellum and no CAT-2 CRM (cross-reacting material) is present in the tissues of this line. Also, unlike most maize lines, CAT-2 cannot be induced in the leaf tissue of A338 upon exposure to light. Finally, a single line (A337), demonstrating a novel catalase developmental program, was identified.     

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated M _r of 124000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.     

Satellite DNA sequences flank amplified DHFR domains in marker chromosomes of mouse fibrosarcoma cells

This study centers on marker chromosomes carrying expanded chromosomal regions which were observed in two independent derivatives of the AA12 murine fibrosarcoma line, the 10^–3 M MTX-res H2 and the 5×10^–7 M MTX-res E. Previous characterization of the marker chromosomes of MTX-res variants showed their common derivation from a marker chromosome (m) of the parental line, endowed with two interstitial C-bands. Cytogenetic evidence pointed to one C-band ofm as the site involved in the chromosomal rearrangements leading to the HSR/ASR chromosomes. ISH of a³H-labeled satellite DNA probe allowed satellite sequences flanking the HSR/ASR in the marker chromosomes, where the C-band was no longer visible, to be detected. FISH experiments using biotinylated DHFR and satellite DNA probes showed that the respective target sequences are contiguous in new marker chromosomes. They also allowed inter- and intrachromosomal rearrangements to be seen at DHFR amplicons and satellite sequences. Double-color FISH using digoxygenated satellite DNA and biotinylated pDHFR7 showed that in a marker chromosome from the H2 cell line the two target sequences are not only adjacent, but closer than 3 Mb, as indicated by overlapping of the different fluorescence signals given by the two probes. Another marker chromosome in the E variant was shown to display a mixed ladder structure consisting of a head-to-head tandem of irregularly-sized satellite DNA blocks, with two symmetrical interspersed DHFR clusters.Abbreviations DHFR dihydrofolate reductase - MTX Methotrexate - HSR Homogeneously Staining Region - ASR Abnormally Staining Region - DM Double Minute - ISH In Situ Hybridization - FISH FluorescenceIn Situ Hybridization     

Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype

The relationship between sequence variation and phenotype is poorly understood. Here, we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in dihydrofolate reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis–Menten‐like in vitro activity curve of thymidylate kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis–Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system‐level implications for bacterial phenotype.     

Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis

A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme showed the maximal caseinolytic activity higher than pH 12.6 in KCl–NaOH buffer at 40°C. Hydrolysis of the oxidized insulin B-chain followed by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus 97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported from a strict anaerobe in this family.