Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Sequences of the cyanogen bromide peptides and complete sequences of the enzyme.

The complete amino acid sequence of dihydrofolate reductase from an amethopterin-resistant strain of Lactobacillus casei has been determined by sequence analysis of peptides produced by cleavage with cyanogen bromide, trypsin, staphylococcal protease, and myxobacter protease. Comparison of this sequence with those of reductases from other bacterial sources shows that the enzymes are homologous. The Lactobacillus casei reductase sequences shows a 29% sequence identity with that of the Escherichia coli enzyme and a 34% identity with the sequence of the enzyme from Streptococcus faecium. The NH2-terminal 68 residues of the L. casei reductase show a 54% sequence identity with that of the enzyme from S. faecium.     

The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67.

The amino acid sequence of a trimethoprim-resistant dihydrofolate reductase (EC 1.5.1.3) specified by the R-plasmid R67 is described. The sequence was deduced from automatic and manual sequence analysis of the intact protein, the fragments produced by cyanogen bromide cleavage, and peptides derived from the largest cyanogen bromide fragment by digestion with trypsin, Staphylococcus aureus V8 proteus, chymotrypsin, and Lysobacter enzymogenes alpha-lytic protease. The complete sequence comprises 78 residues in a single polypeptide chain of molecular weight 8444. No evidence of heterogeneity was obtained, indicating that all subunits of the native enzyme are identical. Comparison of the sequence with that of all known dihydrofolate reductases shows no significant sequence homology.     

Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site

Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements. The effects of ligands of dihydrofolate reductase on the reaction of E. coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase. The E. coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim. Methotrexate was studied in detail and proved to bind to an ATP binding site of the E. coli B enzyme with K1 value of 0.1 mM. The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase. These analyses would indicate that the homologous portion of the amino acid sequence of the E. coli B enzyme provides the ATP binding site. This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.     

R plasmid dihydrofolate reductase with a dimeric subunit structure

Dihydrofolate reductase specified by plasmid R483 from a trimethoprim-resistant strain of Escherichia coli has been purified 2,000-fold to homogeneity using dye-ligand chromatography, gel filtration, and polyacrylamide gel electrophoresis. The protein migrated as a single band on nondenaturing polyacrylamide gel electrophoresis and had a specific activity of 250 mumol/mg min(-1). The molecular weight was estimated to be 32,000 by gel filtration and 39,000 by Ferguson analysis of polyacrylamide gel electrophoresis. When subjected to electrophoresis in the presence of sodium dodecyl sulfate, the protein migrated as a single 19,000-molecular weight species, a fact that suggests that the native enzyme is a dimer of similar or identical subunits. Antibody specific for R483-encoded dihydrofolate reductase did not cross-react with dihydrofolate reductase encoded by plasmid R67, T4 phage, E. coli RT500, or mouse L1210 leukemia cells. The amino acid sequence of the first 34 NH2-terminal residues suggests that the R483 plasmid dihydrofolate reductase is more closely related to the chromosomal dihydrofolate reductase than is the enzyme coded by plasmid R67.     

Dihydrofolate reductase. The stereochemistry of inhibitor selectivity

X-ray structural results are reported for 10 triazine and pyrimidine inhibitors of dihydrofolate reductase, each one studied as a ternary complex with NADPH and chicken dihydrofolate reductase. Analysis of these data and comparison with structural results from the preceding paper (Matthews, D.A., Bolin, J.T., Burridge, J.M., Filman, D.J., Volz, K.W., Kaufman, B. T., Beddell, C.R., Champness, J.N., Stammers, D.K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391) in which we contrasted binding of the antibiotic trimethoprim (TMP) to chicken dihydrofolate reductase on the one hand with its binding to Escherichia coli dihydrofolate reductase on the other, permit identification of differences that are important in accounting for TMP's selectivity. The crystallographic evidence strongly suggests that loss of a potential hydrogen bond between the 4-amino group of TMP and the backbone carbonyl of Val-115 when TMP binds to chicken dihydrofolate reductase but not when it binds to the E. coli reductase is the major factor responsible for this drug's more potent inhibition of bacterial dihydrofolate reductase. A key finding of the current study which is important in understanding why TMP binds differently to chicken and E. coli dihydrofolate reductases is that residues on opposite sides of the active-site cleft in chicken dihydrofolate reductase are about 1.5-2.0 A further apart than are structurally equivalent residues in the E. coli enzyme.     

Selenomethionyl dihydrofolate reductase from Escherichia coli. Comparative biochemistry and 77Se nuclear magnetic resonance spectroscopy.

The biosynthetic replacement of Met residues by selenomethionine (SeMet) facilitates the determination of three-dimensional structure by multiwavelength anomalous diffraction (Yang, W., Hendrickson, W. A., Crouch, R.J., and Satow, Y. (1990) Science 249, 1398-1405). In an effort to examine any biochemical effects due to the replacement of Met residues by SeMet, we chose to compare the kinetic and binding properties of selenomethionyl dihydrofolate reductase with those of the wt enzyme. There are 5 Met residues in Escherichia coli dihydrofolate reductase with 2 located in the Met-20 loop, which is a sequence of residues forming a lid over the active site. Utilizing plasmid pWT8, which affords 10-15% soluble protein as E. coli dihydrofolate reductase, we readily isolated both the SeMet and wt enzymes from E. coli DL41 utilizing a novel purification protocol. Both enzymes exhibited essentially the same kinetic and binding properties, including specific activities (45 mumol/min/mg), Km (7,8-dihydrofolate = 0.39 microM; NADPH = 2.0 microM), kcat (13.5/s), and 1:1 noncovalent inhibitory binding ratios with methotrexate. The inhibitory effects of divalent and monovalent cations on activity were also assessed, with the SeMet-containing enzyme exhibiting a uniformly greater sensitivity than the wt enzyme. We conclude that the biochemical properties of dihydrofolate reductase are virtually unperturbed by SeMet inclusion. Analysis of SeMet dihydrofolate reductase by 77Se nuclear magnetic resonance spectroscopy revealed five distinct resonances, thus indicating the potential value of this technique in employing selenium as a nonperturbing NMR probe of protein structure and function.     

Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus

A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH(Tt)] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH(Tt) appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.     

Sequence analysis of bovine lens aldose reductase

The covalent structure of bovine lens aldose reductase (alditol-NADP+ oxidoreductase, EC 1.1.1.21) was determined by sequence analysis of peptides generated by specific and chemical cleavage of the homogeneous apoenzyme. Peptides, purified by reverse-phase high performance liquid chromatography were subjected to compositional analysis and sequencing by gas-phase automated Edman degradation. Aldose reductase was found to contain 315 amino acid residues. The enzyme is blocked at the amino terminus, and mass spectrometry was employed to identify the blocking acetyl group and to sequence the amino-terminal tryptic peptide. The aldose reductase was shown to contain no carbohydrate despite the fact that the enzyme contains the consensus sequence -Asn-Lys-Thr- for N-linked glycosylation. Comparative sequence analysis and application of algorithms for prediction of secondary structure and nucleotide binding domains are consistent with the view that aldose reductase is a double-domain protein with a beta-alpha-beta secondary structural organization. The NADPH binding site appears to be associated with the amino-terminal half of the enzyme. Modeling studies based on the tertiary structures of dihydrofolate and glutathione reductases indicate that the NADPH binding site begins at Lys-11 and continues with a beta-alpha-beta fold characteristic of nucleotide binding proteins.     

Characterization of an R-plasmid dihydrofolate reductase with a monomeric structure

A plasmid-encoded dihydrofolate reductase that originated in a clinical isolate of Salmonella typhimurium (phage type 179) moderately resistant to trimethoprim has been isolated and characterized. The dihydrofolate reductase (called type III) was purified to homogeneity using a combination of gel filtration, hydrophobic chromatography, and methotrexate affinity chromatography. Polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions indicated that the enzyme is a 16,900 molecular weight monomeric protein. Kinetic analyses showed that trimethoprim is a relatively tight binding inhibitor (Ki = 19 nM) competitive with dihydrofolate. The enzyme is also extremely sensitive to methotrexate inhibition (Ki = 9 pM) and has a high affinity for dihydrofolate (Km = 0.4 microM). The sequence of the first 20 NH2-terminal residues of the protein shows 50% homology with the trimethoprim-sensitive chromosomal Escherichia coli dihydrofolate reductase and suggests that the two enzymes may be closely related. This is the first example of a plasmid encoding for a monomeric dihydrofolate reductase only moderately resistant to trimethoprim, and a resistance mechanism, dependent in part on the high dihydrofolate affinity of the type III enzyme, is proposed.     

The amino acid sequence of the ribosomal protein S8 of Escherichia coli.

The primary structure of protein S8 from the 30S subunit of Escherichia coli ribosomes has been determined by sequencing the peptides derived from tryptic, chymotryptic, thermolytic and staphylococcal protease digestion of the protein. Protein S8 has 129 amino acid residues which result in a molecular weight of 13996. The N-terminal part of the sequence up to position 68 is in complete agreement with the reported sequence data[1,2]. However, differences exist in the C-terminal half, where an additional hydrophobic tryptic peptide has been found.     

Primary structure of a zinc protease from Bacillus mesentericus strain 76

The amino acid sequence of the neutral zinc protease from Bacillus mesentericus strain 76 (MCP 76) has been determined by using peptides derived from digests with trypsin, chymotrypsin, and cyanogen bromide and from cleavage with o-iodosobenzoic acid. The peptides were purified by means of gel filtration and reversed-phase high-performance liquid chromatography and analyzed by automatic sequencing. The protein contains 300 amino acid residues. It proved to be identical with the neutral protease deduced from the DNA precursor sequence of Bacillus subtilis. The residues for zinc and substrate binding are conserved, whereas the number of calcium binding sites is reduced compared to thermolysin. A classification of the neutral zinc protease is discussed.     

The primary structure of thioredoxin from the filamentous cyanobacterium Anabaena sp. 7119

Thioredoxin from the cyanobacterium Anabaena 7119 serves as electron donor to ribonucleotide reductase and as a protein disulfide reductase. This small, heat-stable protein was found to have structural and functional similarities to thioredoxins from both bacterial and mammalian sources. We here report the complete primary structure of Anabaena thioredoxin. The structure was determined by analysis of peptides obtained after cleavage with cyanogen bromide, Staphylococcus aureus protease, and trypsin. The protein consists of 106 residues with the following amino acid sequence: Ser-Ala-Ala-Ala-Gln-Val-Thr-Asp- Ser-Thr-Phe-Lys-Gln-Glu-Val-Leu-Asp-Ser-Asp-Val-Pro-Val-leu-Val-Asp-Phe- Trp-Ala-Pro-Trp-Cys-Gly-Pro-Cys-Arg-Met-Val-Ala-Pro-Val-Val-Asp-Glu- Ile-Ala-Gln-Gln-Tyr-Glu-Gly-Lys-Ile-Lys-Val-Val-Lys-Val-Asn-Thr-Asp- Glu-Asn-Pro-Gln-Val-Ala-Ser-Gln-Tyr-Gly-Ile-Arg-Ser-Ile-Pro-Thr-Leu- Met-Ile-Phe-Lys-Gly-Gly-Gln-Lys-Val-Asp-Met-Val-Val-Gly-Ala-Val-Pro- Lys-Thr-Thr-Leu-Ser-Gln-Thr-Leu-Glu-Lys-His-Leu. The sequence of Anabaena thioredoxin shows a definite homology to the protein from Escherichia coli, with 49% residue identities occurring in the proteins when aligned at the active site disulfide.     

Effects of conversion of an invariant tryptophan residue to phenylalanine on the function of human dihydrofolate reductase

The binding site residue Trp-24 is conserved in all vertebrate and bacterial dihydrofolate reductases of known sequence. To determine its effects on enzyme properties, a Trp-24 to Phe-24 mutant (W-24-F) of human dihydrofolate reductase has been constructed by oligodeoxynucleotide site-directed mutagenesis. The W-24-F mutant enzyme appears to have a more open or flexible conformation as compared to the wild-type human dihydrofolate reductase on the basis of results of a number of studies. These studies include competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase, thermal stability, and protease susceptibility studies of both mutant W-24-F and wild-type enzymes. It is concluded that Trp-24 is important for maintaining the structural integrity of the native enzymes. Changes in relative fluorescence quantum yield indicate that Trp-24 is buried and its fluorescence quenched relative to the other two tryptophan residues in the wild-type human reductase. Kinetic studies indicate that kcat values for W-24-F are increased in the pH range of 4.5-8.5 with a 5-fold increase at pH 7.5 as compared to the wild-type enzyme. However, the catalytic efficiency of W-24-F decreases rapidly as the pH is increased from 7.5 to 9.5. The Km values for dihydrofolate are also increased for W-24-F in the pH range of 4.5-9.5 with a 30-fold increase at pH 7.5, while the Km value for NADPH increases only ca. 1.4-fold at pH 7.5 as compared to the wild type.(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.     

Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.     

The structure of the mutant dihydrofolate reductase from Streptococcus faecium. Partial sequence and order of the limited tryptic and cyanogen bromide peptides.

The major form of dihydrofolate reductase from a methotrexate-resistant mutant (strain A) of Streptococcus faecium var. Durans has been purified on a large scale. Amino acid analysis of this form of the enzyme (isoenzyme 2) reveals an absence of cystine or cysteine, and sedimentation studies indicate a molecular weight of 20,800. The NH2-terminal sequence was determined by Edman degradation of the intact protein and the COOH terminus by selective tritiation and by carboxypeptidase treatment. After the action of trypsin on the citraconylated protein, seven of the expected nine peptides were purified from the digest, and after cyanogen bromide treatment of the unmodified protein, all seven of the anticipated peptides were isolated. The amino acid composition of all of these peptides has been established as well as their complete or partial sequences. From the results it was possible to order these peptides within the sequence and to establish the sequence of the NH2-terminal 60 residues and the COOH-terminal 11 residues.     

Dihydrofolate reductase of the extremely halophilic archaebacterium Halobacterium volcanii. The enzyme and its coding gene

Halobacterium volcanii mutants that are resistant to the dihydrofolate reductase inhibitor trimethoprim contain DNA sequence amplifications. This paper describes the cloning and nucleic acid sequencing of the amplified DNA sequence of the H. volcanii mutant WR215. This sequence contains an open reading frame that codes for an amino acid sequence that is homologous to the amino acid sequences of dihydrofolate reductases from different sources. As a result of the gene amplification, the trimethoprim-resistant mutant overproduces dihydrofolate reductase. This enzyme was purified to homogeneity using ammonium sulfate-mediated chromatographies. It is shown that the enzyme comprises 5% of the cell protein. The amino acid sequence of the first 15 amino acids of the enzyme fits the coding sequence of the gene. Preliminary biochemical characterization shows that the enzyme is unstable at salt concentrations lower than 2 M and that its activity increases with increase in the KCl or NaCl concentrations.     

Nucleotide sequence of the dihydrofolate reductase gene of Saccharomyces cerevisiae

The nucleotide sequence of a DNA fragment that contained the Saccharomyces cerevisiae gene DFR coding for dihydrofolate reductase (DHFR) was determined. The DHFR was encoded by a 633-bp open reading frame, which specified an Mr24264 protein. The polypeptide was significantly related to the DHFRs of chicken liver and Escherichia coli. The yeast enzyme shared 60 amino acid (aa) residues with the avian enzyme and 51 aa residues with the bacterial enzyme. DHFR was overproduced about 40-fold in S. cerevisiae when the cloned gene was present in the vector YEp24. As isolated from the Saccharomyces library, the DFR gene was not expressed in E. coli. When the gene was present on a 1.8-kb BamHI-SalI fragment subcloned into the E. coli vector, pUC18, weak expression in E. coli was observed.     

Purification, properties and primary structure of thioredoxin from Aspergillus nidulans.

This paper reports the purification and the properties of a thioredoxin from the fungus Aspergillus nidulans. This thioredoxin is an acidic protein which exhibits an unusual fluorescence emission spectrum, characterized by a high contribution of tyrosine residues. Thioredoxin from A. nidulans cannot serve as a substrate for Escherichia coli thioredoxin reductase. Corn NADP-malate dehydrogenase is activated by this thioredoxin in the presence of dithiothreitol, while fructose-1,6-bisphosphatase is not. The amino acid sequence of Aspergillus thioredoxin was determined by automated Edman degradation after cleavage with trypsin, SV8 protease, chymotrypsin and cyanogen bromide. The masses of tryptic peptides were verified by plasma-desorption mass spectrometry. The mass of the protein was determined by electrospray mass spectrometry and shown to be in agreement with the calculated mass derived from the sequence (M(r) = 11,564). Compared to thioredoxins from other sources, the protein from A. nidulans displays a maximal sequence similarity with that from yeast (45%).