Portal de Periódicos da CAPES

The Biotin Connection: Severo Ochoa, Harland Wood, and Feodor Lynen

2004; Elsevier BV; Volume: 279; Issue: 38 Linguagem: Inglês

10.1074/jbc.x400005200

1083-351X

M. Daniel Lane,

Click Chemistry and Applications

Unique circumstances sometimes bring us into contact with individuals who will profoundly influence us, particularly in our formative years. In this article I would like to reflect on the circumstances that brought me into contact with three great biochemists, Severo Ochoa (1Ochoa S. Annu. Rev. Biochem. 1980; 49: 1-30Crossref PubMed Scopus (20) Google Scholar), Harland Wood (2Wood H.G. Annu. Rev. Biochem. 1985; 54: 1-41Crossref PubMed Google Scholar), and Feodor Lynen (3, Lynen, F. B. (1964) Information available on the Nobel Museum Web Site: www.nobel.se/,Google Scholar). Each entered the field by a different route: Ochoa as a physician with an interest in physiology, Wood as a bacteriologist trained at Iowa State University, and Lynen as an organic chemist trained in the German tradition with Nobel Prize winner, Heinrich Wieland. They entered the field of biochemistry in the late 1930s when the race was on to discover new enzymes, cofactors, and metabolic cycles. Hans Krebs had formulated the tricarboxylic acid cycle in 1937 and ornithine cycle (now known as the urea cycle) in 1932, some B vitamins had been found to function as cofactors or prosthetic groups of enzymes, and Rudolf Schoenheimer (Columbia University College of Physicians and Surgeons) had demonstrated the dynamic state of tissue proteins using heavy isotopes of hydrogen and carbon (mid-1930s). This was where the action was and it attracted many of the brightest young minds into the field. This was the arena in which Ochoa, Wood, and Lynen were early participants. Excited by discovery, they transmitted this excitement to their younger colleagues. I was fortunate to have scientific associations and enduring friendships with each of them. My connection developed through the B vitamin, biotin, and its role in the reactions catalyzed by a family of biotin-dependent enzymes, notably carboxylases. The B vitamin, biotin, has an interesting history not familiar to most scientists who now make use of it. Today, this vitamin is widely used along with avidin (or its cousin, strepavidin), the specific biotin-binding protein from egg white, to probe biochemical phenomena. Biotinylation of proteins and nucleotides and the use of avidin to “fish out” or detect these molecules from/in complex mixtures has found great utility. It is a curiosity that nature has brought together within the hen's egg the richest source of biotin in the yolk and in the white, a “toxic” factor, avidin, which when fed to animals causes biotin deficiency. In 1936, Kögl and Tönnis isolated 1.1 mg of biotin from more than 500 pounds of egg yolk. Paul György recognized that the distribution, fractionation behavior, and chemical properties of Kögl's yeast growth factor and the anti-egg white injury factor in egg yolk (then called vitamin H) were similar. When Kögl's pure biotin methyl ester became available it was found to be extremely potent in protecting rats against “egg white (i.e. avidin) injury.” Within a few years Vincent Du Vigneaud and colleagues determined the structure of biotin, which cleared the way for an attack on the role of biotin at the molecular level. By 1950 biotin had been implicated in a number of seemingly unrelated enzymatic processes including the decarboxylation of oxaloacetate and succinate; the “Wood-Werkman reaction” (discovered by Harland Wood (2Wood H.G. Annu. Rev. Biochem. 1985; 54: 1-41Crossref PubMed Google Scholar)), i.e. the carboxylation of pyruvate; the biosynthesis of aspartate; and the biosynthesis of unsaturated fatty acids. Of course, we now know that biotin functions in each of these processes as a mobile “CO2 carrier” bound covalently to a carboxylase. The long sought after link between biotin and enzymatic function was provided by Henry Lardy at the University of Wisconsin. Lardy showed that liver mitochondrial extracts catalyzed the ATP- and divalent cation-dependent carboxylation of propionate (subsequently shown to be propionyl-CoA) to form succinate (4Lardy H.A. Adler J. J. Biol. Chem. 1956; 219: 933-942Abstract Full Text PDF PubMed Google Scholar). Later work in the laboratory of Severo Ochoa found that the initial carboxylation product was methylmalonyl-CoA, an intermediate en route to succinyl-CoA. The connection to biotin was made by Lardy with the finding that the propionate-carboxylating activity was lacking in liver mitochondria from rats made biotin-deficient by being fed egg white, which of course contained avidin (KD(biotin) ∼10–15). Moreover, the failure of mitochondrial extract to catalyze the carboxylation of propionate was quickly cured by injecting the rats with biotin. Upon joining the faculty at Virginia Polytechnic Institute in Blacksburg, Virginia in 1956, I decided to try to determine how propionate is metabolized in the liver. Because of its unique features, I settled on bovine liver as the tissue source of the enzyme system to address this question, propionate being a major hepatic carbon source in ruminants. Unlike carbohydrate digestion by monogastric animals, ruminants digest carbohydrates in the rumen, the large anaerobic fore compartment of their multi-compartmented “stomach.” Virtually all carbohydrate is fermented in the rumen to short chain fatty acids, primarily acetate and propionate. Thus glucose, the major digestion product of carbohydrates in monogastric animals, is not available for absorption in ruminants. Propionate, produced in abundance by fermentation in the rumen, is absorbed directly into the portal system and transported to the liver where it is the major carbon source for gluconeogenesis, the pathway leading to glucose production. My entry into this area coincided with Lardy's report that propionate was somehow carboxylated to form succinate. I recall writing to Henry Lardy, and he referred me to Severo Ochoa at New York University School of Medicine. He knew that Ochoa was working on propionate metabolism and had found that propionyl-CoA first became carboxylated to form methylmalonyl-CoA and then was converted to succinyl-CoA. With some trepidation about competing with the Ochoa laboratory, I decided to forge ahead and purify propionyl-CoA carboxylase from bovine liver mitochondria. For the reasons mentioned above bovine liver turned out to be an excellent source of the enzyme. At that point I wrote to Severo Ochoa, and he generously gave me a status report on their progress and put me in contact with the people in his laboratory (Alisa Tietz, Martin Flavin, and later, Yoshito Kaziro) who were working on the enzyme. This initiated what was to be a long relationship with Severo Ochoa and also his colleague, Yoshito Kaziro (now in Tokyo). About that time I applied to the National Science Foundation for a research grant to support my work on propionate metabolism. The grant proposal was rejected because the reviewers felt that I was really “in over my head” competing with the Ochoa laboratory and also because it had been rumored that his laboratory had already crystallized the enzyme from muscle. I knew that this was not true because in my correspondence with Ochoa he had indicated that the crystals turned out to be pyruvate kinase, not propionyl-CoA carboxylase. After much anguish I wrote to the Head of the National Science Foundation Review Committee, Louis Levin, indicating that the Committee was mistaken: “the carboxylase had not been crystallized” and that I thought it was inappropriate for the National Science Foundation to take a position on a grant application based on the size of the laboratory, rather than the merit of the proposal. A few weeks later I received a letter from Lou Levin indicating that the Study Section had reversed its decision and that the grant would be funded. I doubt seriously if that could happen today. Thus began my independent career in research and a developing relationship with Severo Ochoa. In 1959, a paper by Lynen and Knappe appeared in Angewandte Chemie (5Lynen F. Knappe J. Lorch E. Jutting G. Ringelmann E. Angew. Chem. 1959; 71: 481-486Crossref Google Scholar) (later published in full in Biochemische Zeitschrift (6Knappe J. Ringelmann E. Lynen F. Biochem. Z. 1961; 335: 168-176PubMed Google Scholar)) that created tremendous excitement in my laboratory. The paper described the rather remarkable finding that β-methylcrotonyl-CoA carboxylase, a biotin-dependent carboxylase (involved in leucine catabolism in certain bacteria), catalyzed the ATP-dependent carboxylation of “free” biotin in the absence of its acyl-CoA substrate. The product was shown to be a labile carboxylated biotin derivative, later identified as 1′-N-carboxybiotin. Because biotin was believed to be a prosthetic group covalently bound to the enzyme and because free biotin exhibited an extremely high Km, Lynen proposed that the free biotin had accessed the active site of the carboxylase and by mimicking the biotinyl prosthetic group had gotten carboxylated. Shortly thereafter Don Halenz and I succeeded in purifying a related enzyme, propionyl-CoA carboxylase, from bovine liver mitochondria. After convincing ourselves that it too was a biotin-dependent enzyme, we turned our attention to how the biotinyl group was attached to the carboxylase and what enzymatic reactions were involved in its becoming attached to the carboxylase. Dave Kosow, also in my laboratory at Virginia Tech, had just found that extracts of liver from biotin-deficient rats contained catalytically inactive propionyl-CoA apocarboxylase. Moreover, he demonstrated that a soluble ATP-dependent enzyme system in these extracts from the livers of the biotin-deficient animals catalyzed the covalent attachment of [14C]biotin to the apoenzyme, thereby restoring its ability to carboxylate propionyl-CoA (7Moss J. Lane M.D. Adv. Enzymol. Relat. Areas Mol. Biol. 1971; 35 (a review article): 321-442PubMed Google Scholar). Moreover, Dave Kosow showed (7Moss J. Lane M.D. Adv. Enzymol. Relat. Areas Mol. Biol. 1971; 35 (a review article): 321-442PubMed Google Scholar) that upon treating the 14C-biotinylated carboxylase with Streptomyces griseus protease, biocytin (i.e. ϵ-N-biotinyl-l-lysine) was released. This meant, of course, that the biotin prosthetic group had been linked to propionyl-CoA carboxylase through an amide linkage to a lysyl ϵ-amino group. A few years later it became evident that this long (∼14 Å) side arm facilitates oscillation of the 1′-N-carboxybiotinyl prosthetic group between catalytic centers on the enzyme (7Moss J. Lane M.D. Adv. Enzymol. Relat. Areas Mol. Biol. 1971; 35 (a review article): 321-442PubMed Google Scholar). After completing those experiments I invited Severo Ochoa to visit Virginia Polytechnic Institute and to present two lectures, which he graciously agreed to do. One of these talks dealt with propionyl-CoA carboxylase and the other with the genetic code, the two major projects under way in the laboratory of Ochoa at the time. While he was in Blacksburg Dave and I showed him our results on the site of attachment of biotin to the enzyme. We gave him some of the protease and within a month of his return to New York City he confirmed our findings with the heart propionyl-CoA carboxylase. It was at this point in 1962 that I decided to take a sabbatical leave in Munich with Feodor Lynen (known to his colleagues as “Fitzi”) at the Max-Planck Institüt Für Zellchemie where I could continue the work on the enzymatic mechanism by which biotin became attached to propionyl-CoA carboxylase. Before leaving for Munich Dave Kosow and I developed another more potent apoenzyme system with which to investigate the “biotin loading” reaction. This system made use of Propionibacterium shermanii that expressed huge amounts of methylmalonyl-CoA:pyruvate transcarboxylase, another biotin-dependent enzyme studied extensively by Harland Wood. Moreover, this organism had an absolute requirement for biotin in the growth medium, which when grown at very low levels of biotin produced large amounts of the apotranscarboxylase. The choice of the P. shermanii system turned out to be a good one. It so happened that my stay in Munich coincided with Harland Wood's sabbatical leave in Lynen's Institute. This was a two-fold bonus for me, first because Harland was the world's expert on this enzyme and second because it began a lasting personal relationship with him. He has been a role model for me ever since that period in Munich. Harland (1907–1991) grew up on a farm near Mankato, Minnesota. He entered Macalester College in Minnesota where he majored in chemistry and worked his way through college. While a student at Macalester, he met Milly Davis and in their third year of college they married (in 1929, the year of the stock market crash and beginning of the great depression of the 1930s). In those days this required a meeting (for approval I presume) with the President of the college, who needn't have been concerned as she was at his side for the next 62 years. They were an amazing couple, a cooperative inseparable team. My wife and I shared their friendship for more than 30 years. In 1931, Harland became a graduate student in bacteriology in the laboratory of C. H. Werkman at Iowa State University in Ames, Iowa, where he made a discovery that was so controversial, although correct, that it was questioned by his thesis adviser Werkman as well as by leaders in the field of microbial metabolism including C. B. van Niel. Harland had discovered (2Wood H.G. Annu. Rev. Biochem. 1985; 54: 1-41Crossref PubMed Google Scholar) that heterotrophic organisms, such as the Propionibacteria, were able to fix CO2. Prior to this it was believed that only auxotrophs, i.e. chemosynthetic or photosynthetic auxotrophs, could carry out the net synthesis of organic compounds from CO2. His discovery truly opened the area of enzymatic carboxylation in higher organisms. After completing his Ph.D. degree Harland (Fig. 1) did postdoctoral work at the University of Wisconsin with W. H. Peterson and then returned to Iowa State as a faculty member. Harland was an innovator and an improviser. While at Iowa State he decided to conduct CO2 fixation experiments using 13CO2, but because of World War II restrictions he could not gain access to a mass spectrometer nor could he obtain “heavy” 13CO2. In true Woodsian style, he built his own mass spectrometer and constructed a thermal diffusion column in the Science building at Iowa State College (2Wood H.G. Annu. Rev. Biochem. 1985; 54: 1-41Crossref PubMed Google Scholar). In 1946, Harland became Professor and Director of the Biochemistry Department at Western Reserve (now Case-Western Reserve) University. He ran the most democratic department on record in which faculty salaries were determined by the faculty at a meeting where members voted on one another's salary for the upcoming year! Upon arriving in Munich in August of 1962, I indicated to Lynen that I would like to investigate the P. shermanii “biotin loading” enzyme system, and he agreed with my proposal. Because Harland Wood was already at the Institute, I got his advice on growth conditions and for large scale preparations of the transcarboxylase (actually, the apotranscarboxylase). Both Lynen and Wood were quite enthusiastic about the project. It turned out that by growing P. shermanii in biotin-deficient medium the bacteria produced as much of the apotranscarboxylase as the holotranscarboxylase when the organism was grown on normal/biotin-containing medium. Within a short time I was able to resolve and purify both the apotranscarboxylase and the synthetase that catalyzed loading biotin onto the apoenzyme (7Moss J. Lane M.D. Adv. Enzymol. Relat. Areas Mol. Biol. 1971; 35 (a review article): 321-442PubMed Google Scholar). Dave Young, a postdoctoral fellow who had recently completed his medical training at Duke University, and Karl Rominger, a Ph.D. candidate under Lynen's direction, collaborated with me on these studies. Finally, we proved that the synthetase catalyzed a two-step reaction in which the first step involved the ATP-dependent formation of biotinyl-5′-AMP and pyrophosphate after which the biotinyl group was transferred from the AMP derivative to the appropriate lysyl ϵ-amino group of the apotranscarboxylase. While in the midst of these studies, a controversy developed regarding the site at which biotin became carboxylated during catalysis. It was suggested that HCO–3 became incorporated into the 2′-position of the ureido ring of the covalently bound biotinyl prosthetic group of biotin-dependent enzymes and that the 2′-carbon was then transferred to the acceptor substrate. It was suggested that Lynen's experiments (referred to above) had been done with free biotin and not the biotinyl prosthetic group covalently linked to the carboxylase. Such a mechanism would have necessitated opening and then closing the ureido ring of biotin during the course of the reaction, which to a chemist like Lynen didn't make chemical sense. Moreover, this proposal was inconsistent with the known lability of free 1-N-[14C]carboxybiotin. We knew from my earlier studies that enzyme−14CO2−, presumably enzyme−biotin−14CO2− (prepared by incubating propionyl-CoA carboxylase with H14CO–3 and ATP-Mg2+), was even less stable than free 1-N-carboxy-[ 14CO2−]biotin. so we set out to address the issue head on using propionyl-CoA carboxylase as the source of enzyme−biotin−14CO2−. The previous spring before going to Munich, I had found that enzyme−14CO2− (derived from propionyl-CoA carboxylase) could be stabilized by methylation with diazomethane, i.e. enzyme−14CO2− was labile to acid before but was stable after methylation. Moreover, digestion of methylated enzyme−14CO2− ( enzyme−14CO2−CH3) with S. griseus protease produced a single radioactive derivative, presumably methoxy-[14C]carbonyl-ϵ-N-biotinyl lysine. This product had chromatographic properties similar, but not identical, to ϵ-N-biotinyl lysine. Because I did not have the authentic compound for comparison, these experiments could not be completed at the time. Fortunately, Joachim Knappe, a former member of Lynen's research group now at the University of Heidelberg, had synthesized the derivative and provided Lynen with a sample. Thus, we were able to verify the presumptive identification. This proved that the covalently bound biotinyl prosthetic group, like free biotin, was carboxylated at the 1′-N position (8Lane M.D. Lynen F. Proc. Natl. Acad. Sci. U. S. A. 1963; 49: 379-385Crossref PubMed Scopus (31) Google Scholar). Shortly thereafter, Knappe in Heidelberg and Harland Wood on sabbatical in Lynen's laboratory in Munich showed using a similar approach that the carboxybiotin prosthetic groups of β-methylcrotonyl-CoA and transcarboxylase, respectively, had identical structures (9Lynen F. Biochem. J. 1967; 102: 381-400Crossref PubMed Scopus (184) Google Scholar). Taken together these studies proved unequivocally that the site of carboxylation of biotin was on the 1′-N of the biotinyl prosthetic group. By this point in my sabbatical in Lynen's Institute, I began to recognize certain habits of “the Chief.” For example, he had the habit of working in his office until late in the afternoon. Then, around dusk, i.e. 6:00–6:30 p.m., he would emerge to make “rounds” in the Institute, moving from one bench to the next to survey the day's progress or lack of it. Of course not one of the ∼30 investigators would consider leaving until after he had passed through. He ran a “tight ship”! Fitzi had an uncanny memory and could recall details of experiments done weeks earlier. Lynen (1911–1974) (3, Lynen, F. B. (1964) Information available on the Nobel Museum Web Site: www.nobel.se/,Google Scholar) (Fig. 2) was born and spent his entire life in Munich and environs. He received his doctoral training in organic chemistry at the University of Munich with Heinrich Wieland (Nobel Prize in Chemistry in 1927), graduating in 1937. He then married Wieland's daughter, Eva. He was spared the ravages of World War II because of a serious skiing accident, which left him with a persistent limp. Perhaps Lynen's most important contribution was the discovery of acetyl-CoA, the elusive molecule “active acetate,” sought after by many investigators including Fritz Lipmann, David Nachmansohn, and Severo Ochoa. Ochoa had discovered “condensing enzyme,” now known as citrate synthase, which catalyzed the formation of citrate from “active acetate” and oxaloacetate. These discoveries led to an important collaboration between Lynen and Ochoa in which they proved that citrate synthase used acetyl-CoA, along with oxaloacetate, to form citrate. These findings finally answered the question of how “active acetate” entered the citric acid cycle. In 1964 Lynen received the Nobel Prize (with Konrad Bloch) in Physiology or Medicine for his work on “the mechanism and regulation of cholesterol and fatty acid metabolism.” Lynen had strong connections to the United States. Many Americans came to his Institute to do postdoctoral work or sabbaticals. During the period that Harland Wood and I spent in Munich, the other Americans in the group included Esmond Snell, on sabbatical leave from Berkeley, David Young, Walter Bortz, Dick Himes, Paul Kindel, Martin Stiles, and Ed Wawskiewicz. Although Fitzi Lynen was a hard driving biochemist, he did like to socialize over a beer or a martini. On Friday afternoons Harland would often bring a half-gallon bottle of Gilbey's gin to the Institute and prepare martinis in the second floor laboratory. Shortly after returning from Munich in the Summer of 1963, I received a phone call from Severo Ochoa, who asked if I might be interested in joining the faculty of his department at New York University School of Medicine in New York City. My wife, Pat, and I had some concern about moving from the bucolic setting of Blacksburg, Virginia (where we could see 20 miles from our living room window) to the big city. Nevertheless, we relished the new challenges ahead and were ready for a change in lifestyle. We loved New York City and never regretted having made the decision. Severo Ochoa helped make it worthwhile. Severo Ochoa (1905–1993) (1Ochoa S. Annu. Rev. Biochem. 1980; 49: 1-30Crossref PubMed Scopus (20) Google Scholar, 10Kornberg A. Horecker B.L. Cornudella L. Oró J. Reflections on Biochemistry. Pergamon Press, New York1975: 1-14Google Scholar) (Fig. 3) was born in Luarca, Spain, the youngest of seven children. His father was a lawyer and businessman. He completed his M.D. degree (with honors) at the University of Madrid. Though never having studied with him, he was inspired by Ramón y Cajal, the Spanish neuroanatomist and Nobel Prize winner (1906). Following medical school (1929–1931) Ochoa joined Otto Meyerhof's laboratory in Heidelberg where he worked on muscle glycolysis. His early days in science were marked by the upheavals in Europe leading up to World War II. At the time of the Spanish civil war in 1936, he left Spain for Heidelberg for the second and final time. Then in 1938, because of the turmoil in Germany, he moved to Oxford University in England to work in Professor Rudolph Peter's unit. In 1941 he came to the United States where he joined Carl and Gerty Cori at Washington University in St. Louis. In his comments at the Nobel Prize banquet in 1964, Ochoa spoke of those who had influenced him most. I was deeply influenced by my great predecessor Santiago Ramón y Cajal. I entered Medical School too late to receive his teachings directly but, through his writings and his example he did much to arouse my enthusiasm for biology and crystallize my vocation. Among the great names that adorn the roll of Nobel prize-winners in Medicine is that of Otto Meyerhof, my admired teacher and friend, to whose inspiration, guidance and encouragement I owe so very much. I was very fortunate to have worked also under the guidance of other great scientists and I wish to acknowledge my indebtedness to Sir Rudolph Peters and to Nobel prize winners Carl and Gerty Cori who did so much to add new dimensions to my scientific outlook and enlarge my intellectual experience. The seven years (1964–1970) I spent in Ochoa's department were among the most exciting of my scientific career. It was a small department with only a handful of faculty, which at that time included Charles Weissman, Bob Warner, Bob Chambers, Albrecht Kleinschmidt, and Severo. Upon arriving at New York University Medical School in August of 1962, Severo asked me to give 15 lectures in the first year medical student biochemistry course the next month. This course was Ochoa's pride and joy and he and the faculty attended every lecture. (In retrospect, I feel that this is an excellent way to ensure quality control in teaching.) At the time, however, I hadn't relished the idea of having a Nobel prize winner (1964, with Arthur Kornberg, Ochoa's first postdoctoral fellow) in the audience for the first 15 lectures in my new scientific home. Despite knowing that my first few lectures at New York University were not particularly good, after the lecture Severo put his hand on my shoulder and said, “That was an excellent lecture, Dan.” I knew that it hadn't been, but I did appreciate the encouragement. This was typical of Severo's behavior toward young scientists in whom he had confidence. I suspect that his response reflected the encouragement he had received from his mentors during his development. Every afternoon at 3:00 p.m. we took a break for coffee in the department library where we discussed the latest results of our experiments or a hot new paper. Because the faculty was small, these were informal gatherings, which created a sense of camaraderie. Severo never failed to show up for these sessions. We could always count on Charles Weissman for a good, often slightly “off color” joke. “Have you heard the one about the ——?” Because of his innate ability at story telling, Charles was a favorite lecturer of the medical students. His timing was impeccable. Severo had a princely presence in part because of his carriage, tall stature, and silver hair. At national/international meetings, when he walked into a room he attracted hushed attention. Despite this, he had a warm personality and showed genuine concern for his colleagues, associates, and students. It is natural that we feel a closeness to those to whom we are related through research interests. In Hans Krebs book, Reminiscences and Reflections (11Krebs H.A. Reminiscences and Reflections. Oxford University Press, Oxford, UK1981Google Scholar), he illustrates the scientific genealogy leading to Ochoa. We talk rather loosely these days about “impact factor” (and citation index) in evaluating the worth of one's publications, but it is the excitement and joy of doing science, rather than the recognition itself, that motivates us. Research today moves at great speed. Communication is rapid, publication is rapid, and one is left with the impression that everything of importance was done in the past 10 years. However, science is built stepwise on the shoulders of those who came before us. Little is taught today as to how each of our particular areas of the biological sciences developed. For many students the “important stuff” now goes back into the past for only 7–8 years. Most online scientific journals go back only 7–8 years. Fortunately, the Journal of Biological Chemistry is the exception and is to be commended, because it is online all the way back to the point of its origin in 1905. These Reflections may be a sign of recognition that the history of discovery still has importance. I thank my wife, Pat Lane, who assisted with this article and shared these friendships and experiences with me.