Peter Reichard and the Reduction of Ribonucleosides
Year: 2006
Type: article
Abstract: Fig. 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) Enzymatic Synthesis of Deoxyribonucleotides. I. Formation of Deoxycytidine Diphosphate from Cytidine Diphosphate with Enzymes from Escherichia coli (Reichard, P. (1962) J. Biol. Chem. 237, 3513–3519) Electron Spin Resonance of the Iron-containing Protein B2 from Ribonucleotide Reductase (Ehrenberg, A., and Reichard, P. (1972) J. Biol. Chem. 247, 3485–3488) Activation of the Anaerobic Ribonucleotide Reductase from Escherichia coli by S-adenosylmethionine (Harder, J., Eliasson, R., Pontis, E., Ballinger, M. D., and Reichard, P. (1992) J. Biol. Chem. 267, 25548–25552) Peter Reichard was born in 1925 in Wiener Neustadt, a small town in Austria, about 40 km south of Vienna. With the annexation of Austria by Germany in 1938, Reichard and his family moved to Sweden where he attended a private boarding school near Stockholm. There, he developed an interest in science and upon graduating he decided to become a chemical engineer. However, in order to enter the school of engineering in Stockholm, Reichard needed practical experience so he spent a year working in a factory in the far north of Sweden. He recalls, “I enjoyed neither the long cold winter nor the dull work, which did not live up to my expectations. I decided no more chemistry for me (1Reichard P. To be there when the picture is painted.Annu. Rev. Biochem. 1995; 64: 1-28Crossref PubMed Google Scholar).” Instead, Reichard opted to go to medical school and was accepted by the Karolinska Institute in 1944. The school's introductory laboratory course involved qualitative analyses of mixtures of inorganic salts and it was then that Reichard realized how much he enjoyed “shaking test tubes and the outcome of simple experiments (1Reichard P. To be there when the picture is painted.Annu. Rev. Biochem. 1995; 64: 1-28Crossref PubMed Google Scholar).” Thinking that chemistry was not so bad after all, he began to consider a career in biological research and started to work with Einar Hammarsten. His first assignment with Hammarsten was to assist in the construction of a mass spectrometer. However, after just 1 week he dropped a screwdriver and broke a major glass part of the machine. Not surprisingly, he was discharged from his mass spec building duties. Hammarsten instead put him on a project crystallizing deoxyribonuclease. A subsequent visit by David Shemin steered Reichard's interests toward isolating pure 15N-labeled purines and pyrimidines from small amounts of nucleic acids, and using partition chromatography he was able to separate the four common ribonucleosides. In the fall of 1949 Reichard defended his thesis and received a doctorate in medicine. He spent the next 2 years at Karolinska, finishing medical school and doing research. During this time Reichard worked out a method to prepare 15N-nucleosides from biosynthetically labeled nucleic acids. He then injected the nucleosides into rats as precursors of nucleic acids. These experiments led him to conclude that ribonucleosides were incorporated into both RNA and DNA but deoxyribonucleosides were used exclusively for DNA synthesis. From these results, he proposed the existence of an enzyme that transformed ribose to deoxyribose. After finishing medical school in 1951, Reichard spent a year at Stanford University, doing a postdoctoral fellowship with Hubert Loring. He returned to the Karolinska Institute and was appointed Assistant Professor of Medical Chemistry in 1952. He continued his work on pyrimidine biosynthesis and was reasonably successful. His attempts to advance the ribonucleoside reduction story on the other hand were less fruitful. Incubation of labeled ribonucleotides with extracts from various cells never provided convincing evidence for the formation of deoxyribonucleotides. Suddenly, success arrived one day when Reichard added just the right amount of ATP and Mg2+ to the bacterial extract. He later learned that this delicate balance was due to the fact that the function of ATP is 2-fold: it acts as an allosteric effector for the reductase and it transforms cytidine 5′-phosphate (CMP) to cytidine 5′-diphosphate (CDP), the substrate reduced by the enzyme. Too much ATP causes all the CDP to be transformed into inactive cytidine 5′-triphosphate (CTP). In 1961, Reichard joined the faculty at the University of Uppsala as Professor of Medical Chemistry and continued to study ribonucleoside reduction. Using Escherichia coli, he managed to purify two enzyme fractions, A and B, that participated in the formation of deoxycytidine phosphates from CMP. This is the subject of the first Journal of Biological Chemistry (JBC) Classic reprinted here. Reichard found that Fraction A catalyzed the phosphorylation of cytidine 5′-phosphate (CMP) to cytidine 5′-diphosphate (CDP) whereas Fraction B carried out the reduction of the cytidine diphosphate to deoxycytidine diphosphate (dCDP). The reaction catalyzed by Fraction B required ATP, Mg2+ ions, and reduced lipoic acid. Later, Reichard determined that thioredoxin, rather than lipoic acid, was the hydrogen donor in the reaction. Reichard left Uppsala after 2 years to return to the Karolinska Institute as Professor of Medical Chemistry. Enzyme purification became his main focus, and he eventually purified E. coli ribonucleotide reductase using the reduction of CDP as an assay. He discovered that the enzyme, purified from Fraction B above, consists of two proteins, B1 and B2, which were later renamed R1 and R2. R1 contains two sets of allosteric sites that bind nucleoside triphosphates and is thus involved in regulation of the enzyme. R2 is made of two identical subunits and contains two atoms of iron. Although it was difficult to obtain large amounts of the E. coli reductase, Reichard ultimately arrived at a reproducible method that, after 2 weeks of hard work, gave him several milligrams of R1 and R2. This made structural work on the enzyme possible. He found that R2 gave a characteristic spectrum that suggested that the presence of iron in the enzyme was linked to its activity. Reichard then initiated a collaboration with his next-door neighbor, Anders Ehrenberg, who was a pioneer in the application of electron spin resonance (ESR) spectroscopy to biological problems. This work is the subject of the second JBC Classic reprinted here. Much to their surprise, Ehrenberg and Reichard discovered that R2 contains a signal characteristic of a free radical. The presence of this signal was also linked to the enzymatic activity of R2, suggesting that ribonucleotide reduction proceeds by radical chemistry. Eventually Britt-Marie Sjöberg localized the organic radical to tyrosine 122. In 1971 Reichard became Professor of Biochemistry at Karolinska. In the latter part of his career he began to look at how E. coli synthesizes dNTPs in the absence of oxygen. During anaerobic growth, the bacteria must acquire a different mode of ribonucleotide reduction because oxygen is needed for the generation of the tyrosyl radical. Reichard eventually purified two proteins, dA3 and dA1, involved in the anaerobic reduction of CTP to dCTP. dA3 was the actual anaerobic reductase, and dA1 was later determined to be ferredoxin NADP+ reductase. He found that the enzymes required the presence of S-adenosylmethionine (AdoMet), which was thought to be the precursor of an active radical on the enzyme that fulfilled the function of the tyrosyl radical in the aerobic enzyme. In addition to AdoMet, the reaction required NADPH, dithiothreitol, Mg2+, K+ ions, and a Chelex-treated boiled extract from E. coli, designated RT. Reichard eventually showed that the overall reduction of CTP occurred in two steps, which is the subject of the final JBC Classic reprinted here. His model can be seen in Fig. 2. The first step is the activation of the reductase (dA3) by dA1 and RT. During this step AdoMet is reductively cleaved into methionine and 5′-deoxyadenosine. The second step involves the actual reduction of CTP with dithiothreitol as the hydrogen donor. It was eventually determined that the anaerobic ribonucleotide reductase contains a glycyl radical. More information on the anaerobic ribonucleotide reductase from E. coli can be found in Reichard's JBC Minireview (2Reichard P. The anaerobic ribonucleotide reductase from Escherichia coli.J. Biol. Chem. 1993; 268: 8383-8386Abstract Full Text PDF PubMed Google Scholar). Reichard retired in 1991 and remains at the Karolinska Institute as Professor Emeritus. During his time at Karolinska he was a member of the Nobel Faculty and was heavily involved in the Nobel Committee, as he explains in his JBC Reflections (3Reichard P. Oswald T. Avery and the Nobel Prize in Medicine.J. Biol. Chem. 2002; 277: 13355-13362Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Reichard has received many awards and honors including election to the National Academy of Sciences in 1980. He is also a Foreign Honorary Member of the American Academy of Arts and Sciences. 1Biographical information on Peter Reichard was taken from Ref. 1Reichard P. To be there when the picture is painted.Annu. Rev. Biochem. 1995; 64: 1-28Crossref PubMed Google Scholar. 1Biographical information on Peter Reichard was taken from Ref. 1Reichard P. To be there when the picture is painted.Annu. Rev. Biochem. 1995; 64: 1-28Crossref PubMed Google Scholar.
Source: Journal of Biological Chemistry
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1505146594