To be there when the picture is painted.
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Biochemistry↗
Biomedical subjects
P Reichard
Publications and source records attributed to P Reichard.
A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme.
The nrdA and nrdB genes of Escherichia coli and Salmonella typhimurium encode the R1 and R2 proteins that together form an active class I ribonucleotide reductase. Both organisms contain two additional chromosomal genes, nrdE and nrdF, whose corresponding protein sequences show some homology to the products of the genes nrdA and nrdB. When present on a plasmid, nrdE and nrdF together complement mutations in nrdA or nrdB. We have now obtained in nearly homogeneous form the two proteins encoded by the S. typhimurium nrdE and nrdF genes (R1E and R2F). They correspond to the R1 and R2 proteins. Each protein is a homodimer. Together they catalyze the reduction of CDP to dCDP, using dithiothreitol or reduced glutaredoxin, but not thioredoxin, as an electron donor. CDP reduction is strongly stimulated by low concentrations of dATP, presumably acting as an allosteric effector. Protein R2F contains an antiferromagnetically coupled dinuclear iron center and a tyrosyl free radical. The E. coli and S. typhimurium chromosome thus have maintained the information for a potentially active additional class I ribonucleotide reductase, whose role in vivo is as yet unknown. The allosteric regulation of this enzyme differs from that of the normally expressed reductase.
Allosteric Regulation↗
[Milder late effects of diabetes. Good results with intensified insulin therapy].
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Adult↗
Allosteric control of the substrate specificity of the anaerobic ribonucleotide reductase from Escherichia coli.
The reduction of ribonucleotides is catalyzed by different enzymes in aerobic and anaerobic Escherichia coli, each with a different primary and quaternary structure. Here, we describe the allosteric regulation of the substrate specificity of the anaerobic ribonucleoside triphosphate reductase. The enzyme reduced ribonucleotides at a low basal rate. Reduction was stimulated up to 10-fold by an appropriate modulator (dGTP for ATP reduction, ATP for CTP and UTP reduction, and dTTP for GTP reduction). dGTP and dTTP inhibited the reduction of the "incorrect" substrate; dATP inhibited reduction of all four. From kinetic, effector binding, and competition experiments we conclude that the enzyme has two classes of sites, one that binds ATP and dATP and regulates pyrimidine ribonucleotide reduction ("pyrimidine site"), the other that binds dATP, dGTP, and dTTP and regulates purine ribonucleotide reduction ("purine site"). This model differs slightly from the model for the aerobic reductase, but the physiological consequences remain the same and explain how a single enzyme can provide a balanced supply of the four dNTPs. The similarity of a highly sophisticated control mechanism for the aerobic and anaerobic enzymes suggests that both arose by divergent evolution from a common ancestor, in spite of their different structures.
Adenosine Triphosphate↗
Interactions of 2'-modified azido- and haloanalogs of deoxycytidine 5'-triphosphate with the anaerobic ribonucleotide reductase of Escherichia coli.
The anaerobic Escherichia coli ribonucleotide reductase (class III reductase) responsible for the synthesis of the deoxyribonucleotides required for anaerobic DNA replication contains an oxygen-sensitive glycyl radical (Gly-681) suggesting involvement of radical chemistry in catalysis. The amino acid sequence of this enzyme completely differs from that of earlier described aerobic class I (prototype, aerobic E. coli) and class II (prototype, Lactobacillus leichmanii) reductases that use radical chemistry but employ other means for radical generation. Here, we study the interaction between the anaerobic E. coli reductase with the 5'-triphosphates of 2'-chloro-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine, and 2'-azido-2'-deoxycytidine (N3CTP), which are mechanism-based inhibitors of class I and II reductases and, on interaction with these enzymes, decompose to base, inorganic di(tri)phosphate and 2'-methylene-3(2H)-furanone. Also, with the anaerobic E. coli reductase, the 2'-substituted nucleotides act as mechanism-based inhibitors and decompose. N3CTP scavenges the glycyl radical of the enzyme similar to the interaction of N3CDP with the tyrosyl radical of class I enzymes. However, we found no evidence for a new transient radical species as is the case with class I enzymes. Our results suggest that the chemistry at the nucleotide level for the reduction of ribose by class III enzymes is similar to the chemistry employed by class I and II enzymes.
Anaerobiosis↗
Inhibition of ribonucleotide reductase by 2'-substituted deoxycytidine analogs: possible application in AIDS treatment.
After phosphorylation to the corresponding diphosphates, 2'-azido-2'-deoxycytidine and 2'-difluorocytidine act as powerful inhibitors of ribonucleotide reductase. Phosphorylation requires deoxycytidine kinase, an enzyme with particularly high activity in lymphoid cells. Therefore, the deoxycytidine analogs can be expected to inhibit the reductase with some specificity for the lymphoid system. Pretreatment of human CEM lymphoblasts with the analogs considerably increased the phosphorylation of 3'-deoxy-3'-azidothymidine (AzT). The increased phosphorylation of AzT is caused by a prolongation of the S phase of the cell cycle. Our results suggest the possibility of a combination of 2'-substituted deoxycytidine analogs with AzT in the treatment of AIDS. Gao et al. [Gao, W.-Y., Cara, A., Gallo, R. C. & Lori, F. (1993) Proc. Natl. Acad. Sci. USA 90, 8925-8928] have suggested the use of the ribonucleotide reductase inhibitor hydroxyurea for this purpose, since the resulting decrease in the size of deoxyribonucleotide pools decreases the processivity of the HIV reverse transcriptase. From our results it would appear that the 2'-substituted deoxycytidine analogs might be preferable to hydroxyurea.
Acquired Immunodeficiency Syndrome↗
Effects of mutational loss of nucleoside kinases on deoxyadenosine 5'-phosphate/deoxyadenosine substrate cycle in cultured CEM and V79 cells.
The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP synthesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3% in CEM cells and to 10% in V79 cells. This strongly suggests the presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.
Adamantane↗
A risk-benefit assessment of conventional versus intensive insulin therapy.
Some retrospective nonrandomised or cross-sectional studies have shown that higher blood glucose levels are associated with more pronounced microvascular complications in patients with insulin-dependent diabetes mellitus (IDDM). The prospective randomised studies had, until recently, been less definitive. Intensified treatment, and thus lower blood glucose levels, has led to an initial worsening of retinopathy, but this tendency towards more advanced retinopathy has been transient. Albuminuria and manifest neuropathy have been retarded to some extent. Today, 2 long term randomised studies, the Stockholm study and the Diabetes Control and Complications Trial (DCCT), have proven that a lowering of mean blood glucose levels, measured as a lower glycosylated haemoglobin (HbA1c) value, retards or halts retinopathy, nephropathy and peripheral neuropathy. Intensified treatment, whether performed with multiple injections or insulin pumps, leads to some weight gain and a 3-fold increase in the frequency of severe hypoglycaemic episodes. Hypoglycaemia did not cause long term reduced cognitive function in either study, but was unpleasant to the patients. A great majority of patients in the Stockholm study stated that their well-being had increased while participating in the study. The Stockholm programme required 35 minutes extra per patient per month, and a physician and a nurse could tutor 400 patients. This would bring a significant reduction of serious complications and a gain in terms of patient discomfort and cost. A programme of intensified treatment for IDDM is generally indicated and is possible to carry out.
Blood Glucose↗
Mortality and treatment side-effects during long-term intensified conventional insulin treatment in the Stockholm Diabetes Intervention Study.
Altogether, 102 patients were randomized to intensified conventional treatment (ICT) (n = 48) or standard treatment (ST) (n = 54). After 7.5 years, 89 patients remained, and it was shown that microangiopathy was retarded by the lower blood glucose concentrations seen in the patients in the ICT group. HbA1c was reduced from (means +/- SE) 9.5 +/- 0.2% to 7.1 +/- 0.1% in the ICT group and from 9.4 +/- 0.2% to 8.5 +/- 0.1% in the ST group (P < 0.001). Of the patients, 4 in the ICT group and 3 in the ST group died. Mortality was predicted by albuminuria, the amplitude of the sural nerve action potential, and the test of arm blood flow during contraction of the contralateral hand (sympathetic nerve function) at baseline (P < 0.05). Weight increased by 4.4 +/- 1.1 kg in the ICT group and 1.8 +/- 0.7 kg in the ST group (P = 0.05). Atherosclerosis, measured with digital pulse plethysmography, was approximately the same in the groups at baseline and after five years. In each group, 3 patients had myocardial infarctions, and 2 from each group had ketoacidosis once. There was a mean of 1.1 episodes per patient and per year of serious hypoglycemia in the ICT group and 0.4 episodes per patient and per year in the ST group. No adverse incidents or accidents were observed in either group, and there were no differences between the groups with regard to cognitive function measured with a battery of tests.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Flavodoxin is required for the activation of the anaerobic ribonucleotide reductase.
The inactive anaerobic ribonucleotide reductase from Escherichia coli is transformed by a multienzyme system and S-adenosylmethionine + NADPH into a radical protein that is enzymatically active. One of the activating enzyme components was earlier shown to be ferredoxin (flavodoxin):NADP+ reductase. Here we present evidence that flavodoxin, but not ferredoxin, also is a component of the system. Light reduced deazaflavin can substitute for the flavodoxin system. An additional unidentified low-molecular weight component further stimulates the reaction.
5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus.
BACKGROUND: A cause-and-effect relation between blood glucose concentrations and microvascular complications in patients with insulin-dependent diabetes mellitus has not been established. METHODS: We randomly assigned 102 patients with insulin-dependent diabetes mellitus, nonproliferative retinopathy, normal serum creatinine concentrations, and unsatisfactory blood glucose control to intensified insulin treatment (48 patients) or standard insulin treatment (54 patients). We then evaluated them for microvascular complications after 18 months and 3, 5, and 7.5 years. RESULTS: Mean (+/- SD) glycosylated hemoglobin values were reduced from 9.5 +/- 1.3 percent to 7.1 +/- 0.7 percent in the group receiving intensified treatment and from 9.4 +/- 1.4 percent to 8.5 +/- 0.7 percent in the group receiving standard treatment (P = 0.001). In 12 of the patients receiving intensified treatment (27 percent of those included in the analysis) and 27 of those receiving standard treatment (52 percent), serious retinopathy requiring photocoagulation developed (P = 0.01). Visual acuity decreased in 6 patients receiving intensified treatment (14 percent) and in 18 receiving standard treatment (35 percent) (P = 0.02). Nephropathy (urinary albumin excretion, > 200 micrograms per minute) developed in one patient in the group receiving intensified treatment, as compared with nine patients in the group receiving standard treatment (P = 0.01). No patient in the intensified-treatment group had nephropathy with subnormal glomerular filtration rates, as compared with six patients in the standard-treatment group (P = 0.02). The conduction velocities of the ulnar, tibial, peroneal, and sural nerves decreased significantly more in the standard-treatment group than in the intensified-treatment group. The odds ratio for serious retinopathy was 0.4 (95 percent confidence interval, 0.2 to 1.0; P = 0.04) in the intensified-treatment group as compared with the standard-treatment group. The corresponding odds ratio for nephropathy was 0.1 (95 percent confidence interval, 0 to 0.8; P = 0.04). CONCLUSIONS: Long-term intensified insulin treatment, as compared with standard treatment, retards the development of microvascular complications in patients with insulin-dependent diabetes mellitus.
Adult↗
From RNA to DNA, why so many ribonucleotide reductases?
It is generally accepted that DNA appeared after RNA during the chemical evolution of life. To synthesize DNA, deoxyribonucleotides are required as building blocks. At present, these are formed from the corresponding ribonucleotides through the enzymatic action of ribonucleotide reductases. Three classes of enzymes are present in various organisms. There is little sequence similarity among the three classes of reductases. However, enzymic mechanisms and the allosteric behavior of the enzymes from various organisms are strongly conserved, suggesting that the enzymes might have evolved from a common ancestor, with the class III anaerobic Escherichia coli reductase as its closest relative.
Allosteric Regulation↗
The anaerobic ribonucleotide reductase from Escherichia coli.
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Anaerobiosis↗
An iron-sulfur center and a free radical in the active anaerobic ribonucleotide reductase of Escherichia coli.
Anaerobically grown Escherichia coli contain an oxygen-sensitive ribonucleotide reductase. The enzyme requires anaerobic activation by two E. coli fractions with S-adenosylmethionine, NADPH, dithiothreitol, and KCl. We now find that photochemically reduced deazaflavin can substitute for these two fractions and NADPH. The reductase contained roughly equimolar amounts of iron and sulfide, suggesting the presence of an Fe-S complex. The cluster is characterized by a charge transfer band at 420 nm and a low temperature EPR signal centered at g = 2.01 that is difficult to saturate at 14 K, suggested to be a (3Fe-4S)+ cluster. In five different preparations of essentially protein-pure reductase containing widely different amounts of iron, the catalytic activity correlated well with the iron content. The iron signal disappeared during reductive anaerobic activation, with the appearance of a new EPR signal at g = 2.0033 showing a temperature behavior and microwave power saturability consistent with an organic free radical. The signal disappeared after exposure of the activated enzyme to air. We suggest that activation involves generation of a specific amino acid free radical that is dependent on the reduced Fe-S cluster and S-adenosylmethionine. From other work it appears likely that the free radical is localized on glycine 681 of the polypeptide chain.
Anaerobiosis↗
A possible glycine radical in anaerobic ribonucleotide reductase from Escherichia coli: nucleotide sequence of the cloned nrdD gene.
During anaerobic growth of Escherichia coli an oxygen-sensitive ribonucleoside-triphosphate reductase, different from the aerobic ribonucleoside diphosphate-reductase (EC 1.17.4.1), produces the deoxyribonucleoside triphosphates required for DNA replication. The gene for the anaerobic enzyme has now been cloned and was found to contain a 2136-nucleotide coding region, corresponding to 712 amino acid residues, and an Fnr binding site 228 base pairs upstream of the initiator ATG. The deduced amino acid sequence shows 72% identity to a gene of coliphage T4, sunY, hitherto of unknown function, suggesting that the virus codes for its own anaerobic reductase. The location of an organic free radical formed during activation of the bacterial anaerobic reductase is proposed to be on Gly-681, since the pentapeptide RVCGY at positions 678-682 shows a striking similarity to the C-terminal sequence. RVSGY, of pyruvate formate-lyase. During activation of the anaerobically induced pyruvate formate-lyase, the glycine residue of the pentapeptide becomes an organic radical [Wagner, A. F. V., Frey, M., Neugebauer, F. A., Schäfer, W. & Knappe, J. (1992) Proc. Natl. Acad. Sci. USA 89, 996-1000]. The gene for the anaerobic reductase is located at a position around 96 min on the E. coli genomic map.
Amino Acid Sequence↗
Escherichia coli ferredoxin NADP+ reductase: activation of E. coli anaerobic ribonucleotide reduction, cloning of the gene (fpr), and overexpression of the protein.
A specific ribonucleoside triphosphate reductase is induced in anaerobic Escherichia coli. This enzyme, as isolated, lacks activity in the test tube and can be activated anaerobically with S-adenosylmethionine, NADPH, and two previously uncharacterized E. coli fractions. The gene for one of these, previously named dA1, was cloned and sequenced. We found an open reading frame coding for a polypeptide of 248 amino acid residues, with a molecular weight of 27,645 and with an N-terminal segment identical to that determined by direct Edman degradation. In a Kohara library, the gene hybridized between positions 3590 and 3600 on the physical map of E. coli. The deduced amino acid sequence shows a high extent of sequence identity with that of various ferredoxin (flavodoxin) NADP+ reductases. We therefore conclude that dA1 is identical with E. coli ferredoxin (flavodoxin) NADP+ reductase. Biochemical evidence from a bacterial strain, now constructed and overproducing dA1 activity up to 100-fold, strongly supports this conclusion. The sequence of the gene shows an apparent overlap with the reported sequence of mvrA, previously suggested to be involved in the protection against superoxide (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988). We suggest that a frameshift introduced during isolation or sequencing of mvrA caused an error in the determination of its sequence.
Amino Acid Sequence↗
Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli.
Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta 1) and 74 (beta 2) kDa. Immunological and structural evidence suggests that beta 2 is a degradation product of beta 1 and that the active enzyme is a dimer of beta 1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100 degrees C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).
Aerobiosis↗
Activation of the anaerobic ribonucleotide reductase from Escherichia coli by S-adenosylmethionine.
The anaerobic ribonucleoside triphosphate reductase from Escherichia coli reduces CTP to dCTP in the presence of a second protein, named dA1, and a Chelex-treated boiled extract of the bacteria, named RT. The reaction requires S-adenosylmethionine, NADPH, dithiothreitol, ATP, and Mg2+ and K+ ions. It occurs only under anaerobic conditions. We now show that the overall reaction occurs in two steps. The first is an activation of the reductase by dA1 and RT and requires S-adenosylmethionine, NADPH, dithiothreitol, and possibly K+ ions. In the second step, the activated reductase reduces CTP to dCTP with ATP acting as an allosteric effector. During activation, S-adenosylmethionine is cleaved reductively to methionine + 5'-deoxyadenosine. This step is inhibited strongly by S-adenosylhomocysteine and various chelators. The activation of the anaerobic reductase shows a considerable similarity to that of pyruvate formate-lyase (Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., and Gänzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332-1335).
Anaerobiosis↗