Crystal structure of a phosphatase-resistant mutant of sporulation response regulator Spo0F from Bacillus subtilis.

BACKGROUND: Spo0F, a phosphotransferase containing an aspartyl pocket, is involved in the signaling pathway (phosphorelay) controlling sporulation in Bacillus subtilis. It belongs to the superfamily of bacterial response regulatory proteins, which are activated upon phosphorylation of an invariant aspartate residue. This phosphorylation is carried out in a divalent cation dependent reaction catalyzed by cognate histidine kinases. Knowledge of the Spo0F structure would provide valuable information that would enable the elucidation of its function as a secondary messenger in a system in which a phosphate is donated from Spo0F to Spo0B, the third of four main proteins that constitute the phosphorelay. RESULTS: We have determined the crystal structure of a Rap phosphatase resistant mutant, Spo0F Tyr13-->Ser, at 1.9 A resolution. The structure was solved by single isomorphous replacement and anomalous scattering techniques. The overall structural fold is (beta/alpha)5 and contains a central beta sheet. The active site of the molecule is formed by three aspartate residues and a lysine residue which come together at the C terminus of the beta sheet. The active site accommodates a calcium ion. CONCLUSIONS: The structural analysis reveals that the overall topology and metal-binding coordination at the active site are similar to those of the bacterial chemotaxis response regulator CheY. Structural differences between Spo0F and CheY in the vicinity of the active site provide an insight into how similar molecular scaffolds can be adapted to perform different biological roles by the alteration of only a few amino acid residues. These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger within the phosphorelay system which controls sporulation.

Control of solid tumor metastases with a high-molecular-weight derivative of methotrexate.

Methotrexate (MTX) covalently bound to bovine serum albumin (MTX-BSA), injected ip (10 mg/kg) once every 4 days for a total of 4 doses, was more effective than an equivalent dose of free MTX in reducing the number of metastases observed in female (C57BL/6 X DBA/2)F1 mice bearing the sc implanted Lewis lung carcinoma. Treatment with the high-molecular-weight derivative of MTX in addition caused a decreased rate of growth of the primary tumor and a modest increase in the life-span of the tumor-bearing animal. When tumor-bearing mice were killed after receiving injections of [3H]MTX or [3H]MTX-BSA, no difference in the amount of drug was found at the tumor site after 1 hour; however, after 8 or 24 hours, twice as much radioactivity was found in the tumors of mice treated with carrier-bound drug. Analysis of this radioactivity indicated a ratio of 60--80% carrier-bound to 20--40% free MTX.

1H, 15N, and 13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR.

Spo0F, sporulation stage 0 F protein, a 124-residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high-resolution NMR. The 1H, 15N, and 13C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15N TOCSY-HSQC, 15N NOESY-HSQC, HNCA, and HN(CO)CA. Assignments for many sidechain proton resonances are also reported. The secondary structure, inferred from short- and medium-range NOEs, 3JHN alpha coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (alpha/beta)5 fold. Interestingly, comparison of the secondary structure of Spo0F to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, Eur J Biochem 215:573-585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY. This surface may define a region of binding specificity for response regulators. Magnesium titration of Spo0F, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 +/- 5 mM. Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54.

5-Fluoro-2'deoxyuridine 5'-(p-azidophenyl phosphate), a potential photoaffinity label of thymidylate synthetase.

5-Fluoro-2'-deoxyuridine 5'-(p-azidophenyl phosphate) (1), a potential photoaffinity labeling reagent for thymidylate synthetase from a methotrexate-resistant strain of Lactobacillus casei, has been synthesized and characterized. UV254 irradiation of mixtures of thymidylate synthetase with 1, containing 14C-labeled phenyl and 3H-labeled pyrimidine rings, in the presence of excess 5,10-methylenetetrahydrofolate, the cofactor for the reaction, produced two complexes, separable from the native enzyme by polyacrylamide gel electrophoresis, in which only the 3H-containing moiety was bound to the protein. When mixtures of enzyme and 1 were irradiated in the absence of cofactor, complexes separable from the native enzyme were not observed. However, the 14C-containing component of 1 was now bound to the protein in the absence of the 3H-containing portion. The results are discussed in terms of the topography of the enzyme active site.

Synthesis of a fluorescent derivative of amethopterin,.

Fluorescein isothiocyanate was treated with excess diaminopentane and the remaining unsubstituted amino group of the product was condensed, via a carbodiimide-promoted reaction, with a carboxyl group of amethopterin. The final product, a fluorescent derivative of amethopterin, was isolated by chromatography on AE-cellulose and preparative electrophoresis on polyacrylamide. It was shown to be homogeneous by analytical polyacrylamide electrophoresis and thin-layer chromatography. Proof of structure was provided by elemental analysis, absorbance spectra (at pH 7.0, lambdamax at 495 nm; fluorescence emission at 520 nm), and 1H NMR measurements. The fluorescent derivative of amethopterin inhibited transport of amethopterin into Lactobacillus casei and L1210 cells. It was also a good inhibitor of the L. casei and L1210 dihydrofolate reductases and could be used to provide a fluorescent label for the enzymes during polyacrylamide electrophoresis.

Methotrexate 5-aminoallyl-2'-deoxyuridine 5'-monophosphate: a potential bifunctional inhibitor of thymidylate synthase.

Mercuration of 2'-deoxyuridine 5'-phosphate (dUMP) followed by alkylation with allylamine in the presence of K2PdCl4 afforded the 5-aminoallyl deoxynucleotide, which was isolated by sequential Dowex 50 H+ and DEAE-Sephadex chromatography. Further reaction of the product with the N-hydroxysuccinimide ester of methotrexate (MTX) in dry dimethyl sulfoxide gave an MTX-aminoallyl-dUMP covalent complex separable by DEAE-Sephadex chromatography. Reprecipitation with acid from basic solution offered further purification and the structure was confirmed by elemental analysis, NMR and absorbance spectra. The product was an inhibitor of rat liver dihydrofolate reductase (I50 approximately 250 nM, cf. MTX I50 approximately 60 nM) and Lactobacillus casei thymidylate synthase. With the latter enzyme, inhibition was competitive with both nucleotide and folate substrates (Ki = 2.6 and 3.5 microM, respectively) and partial enzyme-inhibitor binary complex could be detected by gel electrophoresis. Large fluorescence changes were observed on titration of the synthase with MTX-aminoallyl-dUMP and alterations in the UV difference spectra similar to those seen on titration of the enzyme with MTX were also noted. The compound was a poor growth inhibitor for cultured murine L1210 and human CCRF-CEM cell lines, which probably reflects low cellular uptake.

The estimation of dihydropteridine reductase in human blood cells.

Significant homology between dihydropteridine reductase (DHPR) from rat and human sources has been established by the ability of polyclonal antibodies raised to the rat-liver enzyme to detect the human protein in Western blots. The antibody also reacted with a single protein in bovine, dog and porcine kidney extracts, however, only trace reactivity was detected in rabbit. Quantitation of Western blots by soft laser densitometry showed that the response was proportional to total protein present in analyses of both pure rat-liver enzyme samples and crude extracts of rat and human liver. The DHPR contents of human blood cells were analysed by this method and the results compared to levels determined in enzymatic assays. Extracts of platelets and lymphocytes showed good correlation between these two methods, however, granulocytes exhibited high apparent enzyme activity but no DHPR protein detectable in blots. Erythrocyte extracts showed approximately 50% lower DHPR protein levels than predicted by activity measurements. These results are discussed in relation to the accuracy of detecting DHPR deficiencies in humans by enzymatic assay of whole blood samples.

Fluorescent inhibitors of thymidylate synthetase.

Two fluorescent derivatives of 2'-deoxy-5-fluorouridine 5'-p-aminophenyl phosphate were prepared by treatment of this compound with fluorescein isothiocyanate in dimethyl sulfoxide or 5-(dimethylamino)naphthalene sulfonyl chloride in pyridine. The products of the reactions were isolated by diethylaminoethylcellulose chromatography and were shown to be homogeneous by polyacrylamide electrophoresis and TLC. Confirmation of the structure was provided by elemental analysis, absorption and fluorescence spectra, PMR measurements, and liberation of nucleotide upon hydrolysis with snake venom phosphodiesterase. The fluorescent derivatives are good competitive inhibitors (Ki approximately10(-6) M) of thymidylate synthetase from a methotrexate-resistant strain of Lactobacillus casei.

Comparative activity of rat liver dihydrofolate reductase with 7,8-dihydrofolate and other 7,8-dihydropteridines.

The various interactions of rat liver dihydrofolate reductase with two unconjugated 7,8-dihydropteridines, 7,8-dihydrobiopterin and 6-methyl-7,8-dihydropteridine, have been compared with those of 7,8-dihydrofolate and folate. Of particular interest was the reactivity demonstrated by 7,8-dihydrobiopterin because of the potential physiological significance of this reaction both in the regeneration of tetrahydrobiopterin, a cofactor for various biological hydroxylations, and as a step in the biosynthesis of this compound from GTP. Kinetic experiments gave Km values of 0.17, 6.42, and 10.2 microM for 7,8-dihydrofolate, 7,8-dihydrobiopterin, and 6-methyl-7,8-dihydropteridine, respectively, with Vmax = 6.22, 2.39, and 1.54 mumol min-1 mg-1. With folate the enzyme showed high affinity (Km = 0.88 microM) but low Vmax (0.20 mumol min-1 mg-1). The natural cofactor was NADPH and a Km of approximately 0.7 microM was measured with each substrate. The enzyme was activated by both p-hydroxymercuribenzoate and urea when assayed with 7,8-dihydrofolate but was inhibited when 7,8-dihydrobiopterin was the substrate. The pH optimum for dihydrofolate reduction was 4 with enhancement at pH greater than or equal to 5.5 in the presence of 1 M NaCl. Peak activity with 7,8-dihydrobiopterin occurred at pH 4.8; this was shifted to pH 5.3 but was not enhanced by 1 M NaCl. Inhibition with methotrexate was similar whether the enzyme was assayed with either the conjugated or unconjugated 7,8-dihydro derivatives. The rat liver enzyme, highly unstable after purification, was stabilized in the presence of the nonionic detergent, Tween-20 (0.1%); however, the comparative properties toward the conjugated and unconjugated substrates were not altered by this treatment.

An abbreviated synthesis of tetrahydropteridines.

5,6,7,8-Tetrahydrobiopterin, the naturally occurring essential cofactor for the enzymatic hydroxylations of phenylalanine, tyrosine and tryptophan, and its synthetic analog 2-amino-6-methyl-5,6,7,8-tetrahydro-4(3H)-pteridinone, have been synthesized in good yield by the direct hydrogenation of 1-(2-amino-1,6-dihydro-5-nitro-6-oxopyrimidin-4-yl-amino)-1,5-dide oxy-L- erythro-pentulose and 2-amino-6-hydroxy-5-phenylazo-4-pyrimidylamino-acetone, respectively. The reactions were carried out at room temperature in trifluoroacetic acid over a platinum catalyst at 2 atm and the products, each containing a mixture of the two possible C-6 isomers, were isolated by precipitation. The simplicity of the preparative method suggests the procedure may be applied generally to the synthesis of all tetrahydropteridines derived from similar pyrimidine precursors.

Pyridine nucleotide interaction with rat liver dihydropteridine reductase.

The interactions of a homogeneous preparation of rat liver dihydropteridine reductase with NADH, NADPH, NAD+, NADP+, and the 1-N6-ethenoadenine derivative of NAD+ have been investigated by fluorescence titration, circular dichroism, equilibrium dialysis, Sephadex G-25 chromatography, and polyacrylamide gel electrophoresis. The procedures indicate that the dimeric enzyme has a definite preference for NADH, but binds only 1 mol of this nucleotide per mol of enzyme. The binary complex of enzyme with NADH is only partially stable to exhaustive dialysis and gel electrophoresis, where it shows greater mobility (0.26) than the free enzyme (0.21); however, the complex can be isolated by Sephadex G-25 chromatography, and characterized with respect to its absorbance spectrum. No ternary complexes are observed when samples of reductase, preincubated with excess NADH, and either the reaction product, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, or the inhibitor, methotrexate, are subjected to polyacrylamide gel electrophoresis.

Synthesis and efficient isolation procedure for gamma-linked fluorescein methotrexate.

Fluorescein isothiocyanate was reacted in dimethyl sulfoxide with a ten-fold excess of diaminopentane, and the mono-substituted thiourea product was isolated by DEAE-cellulose chromatography, lyophilization and acid precipitation from aqueous base. The dried product was then condensed in dry dimethyl sulfoxide with Methotrexate (MTX) activated by prior incubation (30 min) with 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide hydrochloride, and the reaction products were purified by column chromatography on DEAE-cellulose. Exhaustive elution with 1 M ammonium bicarbonate removed several by-products then finally afforded the exclusively gamma-linked fluorescein--MTX derivative. After lyophilization and acid-base precipitation the compound was obtained in good yield (greater than 40%), was homogeneous by reverse-phase HPLC epsilon 493 (0.1 N NaOH) = 66,000 and was a comparable inhibitor to MTX for rat-liver dihydrofolate reductase.