Lactobacillus leichmannii and Escherichia coli ribonucleotide reductases: chemical and structural similarities.
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Escherichia coli↗
Biomedical subjects
J Stubbe
Publications and source records attributed to J Stubbe.
DNA degradation by bleomycin: evidence for 2'R-proton abstraction and for C-O bond cleavage accompanying base propenal formation.
Reaction of poly(dA-[2'S-3H]dU) with activated bleomycin yields [3H]uracil propenal that completely retains the tritium label. In contrast, we have previously shown that reaction of poly(dA-[2'R-3H]dU) with activated bleomycin affords unlabeled uracil propenal [Wu, J. C., Kozarich, J. W., & Stubbe, J. (1983) J. Biol. Chem. 258, 4694-4697]. We have also prepared both cis- and trans-thymine propenals by chemical synthesis and have observed that the trans isomer is the exclusive product of the bleomycin reaction. Moreover, the cis isomer was found to be stable to the conditions of bleomycin-induced DNA degradation. Taken together, these results establish that the formation of trans-uracil propenal occurs via an anti-elimination mechanism with the stereospecific abstraction of the 2'R proton. The question of phosphodiester bond cleavage during base propenal formation has also been addressed by the analysis of the fate of oxygen-18 in poly(dA-[3'-18O]dT) upon reaction with activated bleomycin. The 5'-monophosphate oligonucleotide ends produced from thymine propenal formation have been converted to inorganic phosphate by the action of alkaline phosphatase, and the phosphate has been analyzed for 18O content by 31P NMR spectroscopy. The oxygen-18 is retained in the inorganic phosphate, establishing that the formation of thymine propenal by activated bleomycin proceeds with C-O bond cleavage at the 3'-position.
Bleomycin↗
Isolation of a multifunctional protein with aminoimidazole ribonucleotide synthetase, glycinamide ribonucleotide synthetase, and glycinamide ribonucleotide transformylase activities: characterization of aminoimidazole ribonucleotide synthetase.
5-Aminoimidazole ribonucleotide (AIR) synthetase, glycinamide ribonucleotide (GAR) synthetase, and GAR transformylase activities from chicken liver exist on a single polypeptide of Mr 110,000 [Daubner, C. S., Schrimsher, J. L., Schendel, F. J., Young, M., Henikoff, S., Patterson, D., Stubbe, J., & Benkovic, S. J. (1985) Biochemistry 24, 7059-7062]. Details of copurification of these three activities through four chromatographic steps are reported. The ratios of these activities remain constant throughout the purification. AIR synthetase has an absolute requirement for K+ for activity and under these conditions has apparent molecular weights of 330,000, determined by Sephadex G-200 chromatography, and 133,000, determined by sucrose density gradient ultracentrifugation. Incubation of 18O-labeled formylglycinamidine ribonucleotide (FGAM) with AIR synthetase results in stoichiometric production of AIR, ADP, and [18O]Pi. NMR spectra of beta-FGAM and beta-AIR are reported.
Acyltransferases↗
Purification and characterization of aminoimidazole ribonucleotide synthetase from Escherichia coli.
Aminoimidazole ribonucleotide (AIR) synthetase has been purified 15-fold to apparent homogeneity from Escherichia coli which contains a multicopy plasmid containing the purM, AIR synthetase, gene. The protein is a dimer composed of two identical subunits of Mr 38,500. The N-terminal sequence, amino acid composition, and steady-state kinetics of the protein have been determined. AIR synthetase has been shown to catalyze the transfer of the formyl oxygen of [18O]formylglycinamide ribonucleotide to Pi.
Amino Acids↗
Substrate specificity of formylglycinamidine synthetase.
Formylglycinamidine ribonucleotide (FGAM) synthetase, which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and ATP to FGAM, ADP, glutamate, and Pi, has been purified to homogeneity (sp act. 0.20 mumol min-1 mg-1) from chicken liver by an alternative procedure to that of Buchanan et al. [Buchanan, J. M., Ohnoki, S., & Hong, B. S. (1978) Methods Enzymol. 51, 193-201] (sp act. 0.12 mumol min-1 mg-1). A variety of new analogues of formylglycinamide ribonucleotide have been prepared in which the formylglycinamide arm (R = CH2NHCHO) has been replaced by R = CH3, CH2OH, CH2Cl, CH2NH3, CH2NHCOCH3, CH2NHCOCH2Cl, CH2NHCO2CH2Ph, and L-CHC-H3NHCHO. These compounds have been characterized by 1H and 13C NMR spectroscopy. With compounds R = CH3, CH2OH, and CH2NHCOCH3 and ATP, in the presence or absence of glutamine, FGAM synthetase catalyzes the production of Pi at 4.5, 48, and 20%, respectively, the rate of production of Pi from formylglycinamide ribonucleotide. Only R = CH2NHCOCH3 causes glutaminase activity as well as ATPase activity and has been shown to be converted to the amidine analogue. Both FGAR (R = CH2NHCHO) and the FGAR analogue (R = CH2NHCHOCH3) in the presence of ATP and FGAM synthetase and in the absence of glutamine form a complex isolable by Sephadex G-50 chromatography. FGAM synthetase is thus highly specific for its formylglycine side chain. [18O]-beta-FGAR was prepared biosynthetically, and FGAM synthetase was shown by 31P NMR spectroscopy to catalyze the transfer of amide 18O to inorganic phosphate.
Adenosine Triphosphatases↗
The mechanism of Lactobacillus leichmannii ribonucleotide reductase. Evidence for 3' carbon-hydrogen bond cleavage and a unique role for coenzyme B12.
Reduction of [3'-3H, U-14C]UTP with ribonucleotide reductase showed isotope effects of 1.6, 1.8, and 1.8 at pH 6.1, 7.3, and 8.3, respectively. Similar studies with [3'-3H, U-14C]ATP gave effects of 2.1, 2.1, 1.7, and 1.9 at pH 5.5, 6.1, 7.3, and 8.3, respectively. During the course of the isotope effect determinations the samples were analyzed for the production of 3H2O. Reduction of [3'-3H]UTP at pH 6.1, 7.3, and 8.3 resulted in 0.08, 0.15, and 0.24% of the total 3H being volatilized at 50% reaction. Reduction of [3'-3H]ATP gave no detectable 3H2O at pH 6.1 or 7.3 and a maximum of 0.02% at pH 8.3. The isotope effects in conjunction with 3H2O production indicate that ribonucleotide reductase catalyzes cleavage of the 3' carbon-hydrogen bond of NTPs during the reduction to dNTPs. The role of the cofactor, adenosylcobalamin (AdoCbl), was also investigated. Incubation of [3'-3H]NTP with prereduced ribonucleotide reductase in the absence of reductant (one-turnover conditions) followed by isolation and analysis of AdoCbl for radioactivity indicated no 3H transfer from substrate to cofactor and no 3H2O production. Similar results were obtained in the presence of DTT (multiple turnover conditions). The role of AdoCbl was further investigated by examination of its potential to mediate hydrogen transfer between two substrates. Incubation of [3'-3H]UTP and unlabeled ATP with ribonucleotide reductase and isolation of the products produced indicated no 3H in dATP. These results allow us to postulate a unique role for AdoCbl in the ribonucleotide reductase reaction, that of a radical chain initiator rather than an intermediary hydrogen transfer agent.
Adenosine Triphosphate↗
Mechanism of ribonucleotide reductase from herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs.
Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of [3'-3H]ADP, [3'-H]UDP, and [5-3H] UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of [3'-3H]ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of [beta-32P]ClUDP with the reductase resulted in the production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase (Harris, G., Ator, M., and Stubbe, J. A. (1984) Biochemistry 23, 5214-5225; Ator, M., and Stubbe, J. A. (1985) Biochemistry 24, 7214-7221).
Adenosine Diphosphate↗
Cloning, overproduction, and purification of the B2 subunit of ribonucleoside-diphosphate reductase.
The nrdB gene, which encodes the B2 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1), was cloned into multicopy plasmid pSPS2. This vector, which contains the pL promoter of bacteriophage lambda and the tetracycline resistance gene of pBR322, was transformed into a lysogenic host with a thermolabile repressor. In the newly constructed strain, subunit B2 constituted approximately 25% of the soluble protein after heat induction, an overproduction of several hundredfold relative to the wild-type strain. Purification to homogeneity of the overproduced protein was accomplished by using DEAE and quaternary aminoethyl ion-exchange resins.
Amino Acid Sequence↗
Mechanism of bleomycin: evidence for a rate-determining 4'-hydrogen abstraction from poly(dA-dU) associated with the formation of both free base and base propenal.
When poly(dA-[4'-3H]dU) was degraded by activated bleomycin under a variety of conditions, 50 +/- 10% of the deoxyuridine residues were converted to uracil and uracil propenal, paralleling observations made with DNA. By manipulation of the concentration of O2 in solution, the relative ratio of uracil propenal to uracil could be varied between 0.03 for anaerobic activation and 7.0 for activation at 3 atm of O2. Tritium selection effects on 4'-hydrogen abstraction were also measured under these conditions and found to range from 7.2 to 12.5. These results strongly suggest that the formation of both uracil and uracil propenal is the consequence of a rate-determining 4'-carbon-hydrogen bond cleavage and of an O2-dependent partitioning of the intermediate produced by this cleavage.
Bleomycin↗
Mechanism of bleomycin: evidence for 4'-ketone formation in poly(dA-dU) associated exclusively with free base release.
Incubation of poly(dA-[3'-3H]dU), poly(dA-[5'-3H]dU), or poly(dA-[5'-3H]dT) under a variety of conditions with activated bleomycin resulted in the production of free nucleic acid base, base propenal, and a small amount of 3H2O. Adjustment of the terminated reaction mixture to pH 10 and incubation at 95 degrees C resulted in a time-dependent increase in 3H2O to an amount equal to the amount of free base. If the terminated reaction mixture was incubated with NaBH4 prior to the heat and alkaline treatment, the release of 3H2O was significantly inhibited. These results are consistent with the generation by activated bleomycin of a 4'-ketone yielding free base, with the exchange of the 3'- and 5'-hydrogens by enolization and with the alkaline-induced strand scission occurring from this intermediate.
Bleomycin↗
A multifunctional protein possessing glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase activities in de novo purine biosynthesis.
Three activities on the pathway of purine biosynthesis de novo in chicken liver, namely, glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase, have been found to reside on the same polypeptide chain. Three diverse purification schemes, utilizing three different affinity resins, give rise to the same protein since the final material has identical specific activities for all three enzymatic reactions and a molecular weight on sodium dodecyl sulfate gels of about 110 000. A single antibody preparation precipitates all three activities and binds to the multifunctional protein obtained by two methods in Western blots. Partial chymotryptic digestion of the purified protein gives rise to two fragments, one possessing glycinamide ribonucleotide synthetase activity and the other containing glycinamide ribonucleotide transformylase activity.
Acyltransferases↗
Mechanism of inactivation of Escherichia coli ribonucleotide reductase by 2'-chloro-2'-deoxyuridine 5'-diphosphate: evidence for generation of a 2'-deoxy-3'-ketonucleotide via a net 1,2 hydrogen shift.
Sodium borohydride or ethanethiol protects the Escherichia coli ribonucleoside-diphosphate reductase (RDPR) from inactivation by 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP). Incubation of [3'-3H]ClUDP with RDPR in the presence of NaBH4 allowed trapping of [3H]-2'-deoxy-3'-ketouridine 5'-diphosphate. Degradation of the reduced ketone by a combination of enzymatic and chemical methods indicated that the hydrogen originally present in the 3'-position of ClUDP is transferred to the beta-face of the 2'-position of 2'-deoxy-3'-keto-UDP. RDPR therefore catalyzes a net 1,2 hydrogen shift. Incubation of RDPR with ClUDP in the presence of ethanethiol allowed trapping of 2-methylene-3(2H)-furanone, the species responsible for inactivation of RDPR. Trapped 2-[(ethylthio)methyl]-3(2H)-furanone was identical by 1H NMR spectroscopy with material synthesized chemically. Both subunits of the enzyme are covalently radiolabeled in the reaction of RDPR with [5'-3H]ClUDP. Studies with [3'-3H]ClUDP and prereduced RDPR in the absence of a reductant and with oxidized RDPR indicated that the redox-active thiols of the B1 subunit are not involved in inactivation of the enzyme by ClUDP.
Borohydrides↗
Identification of two alpha-ketoglutarate-dependent dioxygenases in extracts of Rhodotorula glutinis catalyzing deoxyuridine hydroxylation.
Attempts to isolate deoxyuridine 2'-hydroxylase from Rhodotorula glutinis by the procedure of Warn-Cramer et al. (Warn-Cramer, B. J., Macrander, L. A., and Abbott, M. T. (1983) J. Biol. Chem. 258, 10551-10557) have led to the identification and partial purification of a newly recognized alpha-ketoglutarate-requiring oxygenase. This activity, designated deoxyuridine (uridine) 1'-hydroxylase, in the presence of iron and ascorbate, catalyzes the conversion of deoxyuridine (uridine), O2, and alpha-ketoglutarate to uracil, deoxyribonolactone (ribonolactone), CO2, and succinate. Incubation of [1'-3H]uridine with this activity results in time-dependent formation of uracil concomitant with production of CO2 and 3H2O. No Vmax/Km isotope effect is observed on this reaction. Uracil production is accompanied by stoichiometric production of ribonolactone identified by NMR spectroscopy. Also reported in this paper is the partial purification and characterization of the alpha-ketoglutarate-requiring enzyme, deoxyuridine 2'-hydroxylase. Incubation of [2'-alpha-3H]deoxyuridine with this activity results in concomitant production of uridine and 3H2O. Incubation with [2'-beta-3H] deoxyuridine results in the production of uridine whose specific activity is identical to that of the starting material. This enzyme catalyzes the conversion of deoxyuridine to uridine with retention of configuration. No isotope effect is observed on this transformation.
Deoxyuridine↗
Current ideas on the chemical mechanism of ribonucleotide reductases.
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Binding Sites↗
Mechanism of inactivation of Escherichia coli and Lactobacillus leichmannii ribonucleotide reductases by 2'-chloro-2'-deoxynucleotides: evidence for generation of 2-methylene-3(2H)-furanone.
Incubation of 2'-chloro-2'-deoxy[3'-3H]uridine 5'-diphosphate ([3'-3H]ClUDP) with Escherichia coli ribonucleotide reductase (RDPR) and use of thioredoxin-thioredoxin reductase as reductants result in release of 4.7 equiv of 3H2O/equiv of B1 protomer, concomitant with enzyme inactivation. Inactivation is accompanied by the production of 6 equiv of inorganic pyrophosphate [Stubbe, J. A., & Kozarich, J.W. (1980) J. Am. Chem. Soc. 102, 2505-2507] and by the release of uracil as previously shown [Thelander, L., Larsson, A., Hobbs, J., & Eckstein, F. (1976) J. Biol. Chem. 251, 1398-1405]. Reisolation of RDPR by Sephadex chromatography and analysis by scintillation counting indicate that 0.96 equiv of 3H is bound per protomer of the B1 subunit of the inactivated enzyme. Incubation of [5'-3H]ClUDP with RDPR followed by similar analysis indicates that 4.6 mol of 3H is bound per protomer of the B1 subunit of the inactivated enzyme. No 3H2O is released, and 6 equiv of inorganic pyrophosphate is produced during the inactivation. RDPR is protected against inactivation when dithiothreitol (DTT) is used as a reductant in place of thioredoxin-thioredoxin reductase. Incubation of [5'-3H]ClUDP with RDPR and DTT results in the isolation of CHCl3-extractable material that exhibits infrared absorptions at 1710 and 1762 cm-1. The infrared spectrum and the NMR spectrum of the CHCl3-extracted material are very similar to model compounds prepared by the interaction of 2-methylene-3(2H)-furanone with ethanethiol. Incubation of ribonucleoside-triphosphate reductase (RTPR) from Lactobacillus leichmannii with [3'-3H]ClUTP and 3 mM DTT also results in time-dependent 3H2O release concomitant with enzyme inactivation. Reisolation of the inactive protein by Sephadex chromatography followed by radiochemical analysis indicates that 0.4 equiv of 3H is bound covalently per mol of inactivated enzyme. Similar studies with [5'-3H]ClUTP indicate that 2.9 equiv of 3H is bound covalently per mol of inactivated enzyme. No 3H2O is released. High concentrations of DTT protect the enzyme against inactivation. Extraction of the enzymatic reaction mixture with CHCl3 and analysis of the isolated products result in an infrared spectrum and an NMR spectrum remarkably similar to those observed with the E. coli RDPR. Data presented are consistent with the proposal that both the E. coli and L. leichmannii enzymes are able to catalyze the breakdown of the appropriate 2'-chloro-2'-deoxynucleotide to a 3'-keto-2'-deoxynucleotide that can collapse to form the reactive sugar intermediate 2-methylene-3(2H)-furanone.(ABSTRACT TRUNCATED AT 400 WORDS)
Deoxyuracil Nucleotides↗
The mechanism of free base formation from DNA by bleomycin. A proposal based on site specific tritium release from Poly(dA.dU).
Poly(dA.dU), which is specifically tritiated at the 1'-, 2'- (ribo configuration), 3'-, or 4'-position of deoxyuridine, has been synthesized and the fate of the tritium has been determined upon degradation of the polymer by bleomycin, Fe(II), and O2. No tritium is labilized from the 1'-3H-labeled polymer as 3H2O; however, the resulting 3-(uridin-1'-yl)-2-propenal (uracil propenal) has the expected specific activity. The 2'-3H-labeled polymer affords 3H2O and no label in the uracil propenal. This result and the lack of solvent incorporation into the uracil propenal suggest that proton abstraction from C-2' to afford the trans-propenal is highly stereospecific. For the 3'-3H-labeled polymer, 3H2O is formed and the specific activity of the uracil propenal is identical to that of the deoxyuridine. This suggests that the labilization of the 3'-H is exclusively associated with free uracil formation. 3H2O is also formed from the 4'-3H-labeled polymer. These findings along with previous studies are consistent with the formation of uracil propenal and free uracil by the trapping of the initially formed 4'-radical species by O2 or by a monooxygen species, respectively.
Aldehydes↗
Interaction of 3'-[3H]2'-Chloro-2'-deoxyuridine 5'-triphosphate with ribonucleotide reductase from Lactobacillus leichmannii.
Incubation of [3'-3H]2'-chloro-2'-deoxyuridine 5'-triphosphate (CldUTP) with adenosylcobalamin (AdoCbl), reductant, and ribonucleotide reductase from Lactobacillus leichmannii results in the production of 3H2O, uracil, tripolyphosphate, 5'-deoxyadenosine, and cob(II)alamin. The rate of 3H2O release (0.19 mumol/min/mg) is almost identical with the rate of UTP reduction (0.24 mumol/min/mg). The amount of 3H2O release is dependent upon the enzyme to cofactor ratio. With a ribonucleotide reductase: AdoCbl ratio of 1:1000, approximately 500 eq of 3H2O are released. At this time the enzyme is still active, but further destruction of cofactor and turnover of CldUTP is prevented by competitive inhibition of Co(II) + 5'-deoxyadenosine with AdoCbl for binding to ribonucleotide reductase. The 5'-deoxyadenosine and AdoCbl reisolated during incubation of [3'-3H]CldUTP and ribonucleotide reductase contains no detectable radioactivity.
Cobamides↗
Mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Evidence for 3'-C--H bond cleavage.
Incubation of the pyrimidine [3'-3H]UDP with ribonucleotide reductase resulted in an isotope effect on the conversion to dUDP which varied as a function of pH and allosteric effectors (pH, kH/kT, effector): 6.6, 4.7, ATP; 7.6, 3.3, ATP; 7.6, 2.6, dATP; 7.6, 2.0, TTP; 8.4, 2.8, ATP. During this reaction 3H2O was also released. The lower the pH of the reaction, the larger the isotope effect, and the smaller the amount of 3H2O produced. At 50% conversion of UDP to dUDP and at pH 7.6, approximately 0.5% of total 3H present in solution was volatilized, while at pH 8.4, approximately 0.9% was volatilized. Similar experiments in which the purine [3'-3H]ADP was incubated with ribonucleotide reductase also resulted in an isotope effect on its conversion to dATP which varied as a function of pH (pH, kH/kT with dGTP as an effector); 6.6, 1.9; 7.6, 1.7; 8.6, 1.4. Furthermore, 3H2O was also released as a function of the extent of the reaction. At 50% turnover and pH 7.6, approximately 0.6% of 3H2O was volatilized, while at pH 8.6 approximately 1.25% was released. Two control experiments in which either the B1 subunit of ribonucleotide reductase was inactivated with 2'-chloro-2'-deoxyuridine 5'-diphosphate or the B2 subunit of ribonucleotide reductase was inactivated with 2'-azido-2'-deoxyuridine 5'-diphosphate and then the enzyme incubated with [3'-3H]ADP or [3'-3H]UDP indicated that in neither case was 3H released. Both B1 and B2 subunits are required for cleavage of the 3'-C--H bond. Incubation of [3'-3H]dADP or [3'-3H]dUDP with ribonucleotide reductase produced no measurable release of 3H. These data clearly indicate that conversion of a purine or pyrimidine diphosphate to a deoxynucleotide diphosphate by Escherichia coli ribonucleotide reductase requires cleavage of the 3'-C--H bond of the substrate. The fate of the 3'-H of the substrate was also determined. Incubation of [3'-2H]UDP with ribonucleotide reductase resulted in the production of [3'-2H]dUDP.
Allosteric Regulation↗