Konrad Bloch—A Pioneer in Cholesterol and Fatty Acid Biosynthesis

Konrad Bloch—A Pioneer in Cholesterol and Fatty Acid Biosynthesis

Biochemical and Biophysical Research Communications 292, 1117–1120 (2002) doi:10.1006/bbrc.2002.2031, available online at http://www.idealibrary.com o...

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Biochemical and Biophysical Research Communications 292, 1117–1120 (2002) doi:10.1006/bbrc.2002.2031, available online at http://www.idealibrary.com on

Konrad Bloch—A Pioneer in Cholesterol and Fatty Acid Biosynthesis Dennis E. Vance* ,1 and Howard Goldfine† *Department of Biochemistry and Canadian Institutes of Health Research Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada; and †Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

EARLY YEARS Konrad Emil Bloch was born in Neisse, Germany, on January 21, 1912. His father’s name was Frederick Bloch and progenitors had lived in this town in Upper Silesia for 3 or 4 generations (1). Although Frederick Bloch had a law degree, he took over the family business of making draperies. In 1914, World War I began and Frederick Bloch became an officer. The family survived the war and the business continued. Konrad Bloch obtained his schooling in Neisse, attending an elementary school from the age of 6 to 9 and then high school (humanistic gymnasium) for the next 9 years. His interest in metallurgy led Bloch to attend University at the Technische Hochschule in Munich, Germany. However, it was the organic chemistry lectures of Hans Fischer that led Bloch into chemistry (2). In Munich, Bloch also regularly attended the Munich Chemical Society, where he was greatly influenced by lectures by Richard Willsta¨tter, Heinrich Wieland, and Alfred Windaus. In 1934, the Dean of the Technische Hochschule informed Bloch that he was ineligible to continue because Professor Fischer would not have him as a graduate student (2). This was not true, but simply the beginning of the Nazi policy to eliminate Jews from German society. In fact, Fischer wrote a letter of recommendation for Bloch that stated only, “Herr Bloch ist gut” (1). Fischer arranged for Bloch to become a research assistant at the Schweizerisches Ho¨henforschung’s Institut in Davos. Bloch’s first assignment was to determine if cholesterol was among the lipids of human tubercle bacilli. Bloch’s experiment clearly showed that cholesterol was absent, confirming an earlier report from Erwin Chargaff. Bloch next investigated, and found, phosphatidic acid in the tubercle bacillus, which contradicted a report from R. J. Anderson at Yale University (2). About this time Bloch’s permit in Switzerland was about to expire and Bloch wrote to Anderson to see if he could join his laboratory. Bloch subsequently received a letter from the Dean 1

To whom correspondence should be addressed. Fax: (780) 4923383. E-mail: [email protected]

indicating that he had been accepted as an assistant in biological chemistry. However, a second letter from Anderson indicated there was not a stipend associated with the position. Nevertheless, Bloch moved to the United States in part because his future wife, Lore, whom he had met in Munich, had moved to America (1). Bloch arrived in the United States in December 1936. Anderson did not think that Bloch should come to Yale because he believed Bloch might learn more from Hans Clarke at Columbia University. Clarke was prepared to give Bloch credit for his earlier work in Davos toward a Ph.D. degree but required that he do some lab work at Columbia, where he synthesized a series of N-alkylcysteine derivatives (3). After obtaining his Ph.D., Bloch became a postdoctoral fellow with Rudolf Schoenheimer, who had joined Clarke’s department and was introducing the use of stable isotopes to trace metabolic pathways. Bloch stated in a later interview that “Schoenheimer was first reluctant to hire me because he didn’t think my Ph.D. thesis was very impressive” (1). This reservation must have been short-lived because Bloch and Schoenheimer published a number of papers on creatine metabolism in the Journal of Biological Chemistry. During his time with Schoenheimer, Bloch also started to work on the origin of the oxygen atom in cholesterol, but without success (2). In 1941, tragically Schoenheimer committed suicide and the work in his laboratory was divided among three postdoctoral fellows, David Rittenberg, David Shemin, and Konrad Bloch. Bloch ended up with the cholesterol problem, Shemin with amino acid and heme metabolism, and Rittenberg with protein synthesis and turnover (2). The division of projects occurred by drawing lots (1, 2). The “luck of the draw” led Konrad Bloch into lipid metabolism. THE PATHWAY OF CHOLESTEROL BIOSYNTHESIS The structure of cholesterol was solved in 1932 by Heinrich Wieland and H. Dane (4). At that time, no

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clues in the structure led to reasonable predictions about the biosynthetic pathway. Major insight came from a paper published in 1937 by Sonderhoff and Thomas (5) that arrived in the United States only in 1941 because of the war (2). This paper reported very high incorporation of deuterium-labeled acetate into the sterol fraction of yeast. Thus, Bloch and Rittenberg fed deuterium-labeled acetate to rats and mice and found substantial incorporation of label into cholesterol and fatty acids (6, 7). Subsequent studies added to the knowledge that acetate was a precursor of cholesterol and that cholesterol was a precursor of bile acids and steroid hormones. In 1946, Bloch was enticed by a former graduate student of Schoenheimer’s and a friend, Earl Evans, to move to the Department of Biochemistry at the University of Chicago. In Chicago, in addition to studies on cholesterol biosynthesis, Bloch worked on the biosynthesis of glutathione as a model system for protein biosynthesis. His work showed that amino acids, ATP, and two specific enzymes were required for synthesis of the tripeptide. From studies in other labs, it became apparent that glutathione was not a good model of protein synthesis and Bloch abandoned this line of research. For many years, the Bloch lab worked on the origin of the 27 carbons of cholesterol in competition with the laboratories of John Cornforth and George Popja´ k. Through the efforts of these labs, it was demonstrated that all 27 carbons were specifically derived from either the methyl or the carboxyl carbon of acetate (2, 8). In 1934, the British chemist Sir Robert Robinson had proposed that squalene might be folded into cholesterol. It was only in 1952 that R. G. Langdon and Bloch demonstrated that squalene was indeed a precursor of cholesterol (9). Although Robinson had an important insight, the proposed folding was not correct. The correct folding of squalene into lanosterol was predicted by the organic chemist R. B. Woodward and Bloch in 1953 (10). The conversion of squalene to lanosterol involves the migration of methyl groups (from C 8 to C 14 and from C 14 to C 13). The migration of the methyl groups was described independently by Bloch’s lab (11) and by Cornforth and Popja´ k (12). Missing links in the scheme between acetate and squalene were 5-, 10-, and 15-carbon intermediates. An important clue to the identity of these intermediates came from studies in Merck, Sharp and Dohme laboratories that resulted in the isolation of mevalonic acid and the demonstration that it could substitute for acetate in acetate-requiring strains of Lactobacillus acidophilus (13). Subsequently, others showed mevalonic acid to be a precursor of squalene and sterol (14). Bloch’s group, Fyodor Lynen’s group in Germany, and Cornforth and Popja´ k in England were able to elucidate which isoprene phosphates were derived from me-

valonate and were intermediates in the conversion of acetate to squalene (15). The final part of the puzzle was to determine the pathway by which lanosterol was converted to cholesterol. This conversion involves three demethylation reactions and hydrogenation of double bonds. Once again Bloch’s laboratory was heavily involved in these studies (15). The key role Konrad Bloch played in elucidating the pathway for cholesterol biosynthesis was the basis for the Nobel Prize in Physiology or Medicine that was awarded to him and Lynen in 1964. In an interview in 1993, Bloch was asked if he had expected to receive the prize (1). He replied: No. I think I can truthfully say that I thought someone with my interests and my position had a finite chance, but no more. It was not a thought that preoccupied me nor was it an incentive. It came as a total surprise. I was fifty-two, and what I set out to do was by no means complete. I think the award was for the cumulative impact of research, not only on cholesterol biosynthesis, but also fatty acid synthesis. That is what the citation says.

FATTY ACID BIOSYNTHESIS As indicated by the Nobel citation, Bloch had a longstanding and parallel interest in fatty acid biosynthesis. In a classic experiment, Bloch and Rittenberg demonstrated in 1944 that fatty acids were formed from acetate in animals (7). His interest in fatty acid metabolism was renewed when T. T. Chen and Bloch reinvestigated the origin of the oxygen atom of cholesterol and demonstrated (15 years after Bloch first attempted this experiment with Schoenheimer) that it originated from O 2 (2). This result stimulated his interest in “oxygen as an essential biosynthetic reagent, and led me to investigate some consequences for aerobic versus anaerobic patterns of metabolism” (2). In 1960, Bloomfield and Bloch demonstrated the oxygen-dependent desaturation of long-chain acyl-CoA derivatives in a yeast extract (16). The requirement for NADH was also shown and later Schroepfer and Bloch showed that the removal of the two hydrogens of stearic acid to yield oleic acid was stereospecific (17). These findings led Bloch to think about the mechanism of unsaturated fatty acid synthesis in anaerobic organisms that could not use an oxygen-dependent mechanism. In a series of studies, Bloch’s laboratory showed that anaerobic and other bacteria produced unsaturated fatty acids by a novel pathway, not used by eukaryotic organisms, in which the double bond was introduced during the process of chain elongation (18). These studies explained the presence of the 18-carbon 11-cis-vaccenic acid in bacteria, instead of oleic acid, found in eukaryotes, that has the double bond in the ⌬ 9 position. Bloch and co-workers isolated the enzyme required for this step, ␤-hydroxydecanoyl thioester dehydrase, and found that the reaction required a heat-

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stable protein (19) with properties similar to those of a protein shown by Roy Vagelos and co-workers to be required for fatty acid synthesis in Clostridium kluyveri (20). The two groups met at the 1961 meeting of the Federation of American Societies for Experimental Biology in Atlantic City and agreed “not to compete but to pursue their separate objectives that had led to the chance discovery of acyl-carrier protein. Roy’s laboratory was primarily interested in the mechanism of chain elongation, while we wanted to know how unsaturated fatty acids are formed anaerobically” (2). Subsequent studies demonstrated the key role for ␤-hydroxydecanoyl thioester dehydrase in the biosynthesis of unsaturated fatty acids in E. coli. Work on the mechanism of unsaturated fatty acid synthesis in bacteria led to the development of an acetylenic substrate that was converted to an allene by the dehydrase. This allene rapidly reacted with, and inactivated, the dehydrase (2). This acetylenic “suicide” substrate of the dehydrase opened a major area of research on the use of “suicide” substrates in enzymology and pharmacology. Bloch considered the name “suicide” to be a misnomer since “this anthropomorphological term connotes a deliberate act on the part of the enzyme, but this is not strictly the case. Dehydrase falls prey to trickery because it fails to distinguish between the C ␣ protons of the acetylenic and olefinic substrates, an error that seals its fate” (2). He thought suicide inhibition should be more correctly called mechanism-based enzyme inactivation. Bloch’s thinking and interests were also colored by a deep curiosity in evolutionary mechanisms (see the article by Harold White (20a) in this issue). Thus, Bloch initiated studies in the late 1960s on the enzymatic synthesis of fatty acids in mycobacteria, which, like eukaryotic organisms, possess a very large, multifunctional fatty acid synthetase in which seven enzymatic activities of fatty acid synthesis reside in a single polypeptide chain (21). The active form of the synthetase is a dimer of this polypeptide. In E. coli and most other bacteria, the individual enzymes of fatty acid synthesis reside on separate polypeptides. In the course of these studies, a heat-stable factor that stimulated the reaction many-fold was identified (22). Subsequently, this factor was shown to be a polysaccharide containing 3-O-methylmannose (23). The polysaccharide binds long-chain C 22 and C 24 fatty acids uniquely made by the mycobacterial fatty acid synthetase and relieves feedback inhibition of the enzyme by the fatty acid product (21, 23–25). EVOLUTION AND THE STRUCTURE OF CHOLESTEROL Bloch’s evolutionary interest brought him back to cholesterol as a major focus in the late 1970s: why had

the structure of cholesterol evolved as such? The immediate product of squalene cyclization is lanosterol. Is there an important evolutionary advantage for lanosterol to undergo the many subsequent reactions to yield cholesterol? What was the driving force for the formation of cholesterol once the presence of oxygen permitted the biosynthesis of lanosterol (26)? Subsequent work emanating from Bloch’s lab showed that indeed, in several instances, cholesterol would function whereas lanosterol could not replace cholesterol (27). For example, yeast is a sterol auxotroph when grown anaerobically. Cholesterol will satisfy this sterol requirement whereas lanosterol will not. Insects do not have the ability to make sterols and require dietary cholesterol. Lanosterol will not substitute for cholesterol (27). The interactions of sterols with phospholipids also showed that cholesterol was superior to lanosterol. Investigations into the leaking of glucose from phosphatidylcholine vesicles demonstrated that cholesterol, but not lanosterol, impeded this process (27). The microviscosity of phosphatidylcholine vesicles was four times higher with cholesterol than with lanosterol. Moreover, the partially demethylated intermediates were intermediate in their ability to substitute for cholesterol. These and other studies led Bloch to conclude in an article published in 1983 (27): A sterol that fits and interacts effectively with the phospholipid partner will alter the physical state of the membrane more than a molecule that is poorly accommodated. Lanosterol, the earliest intermediate in the sterol pathway fits least and cholesterol, the end product, fits best. The fitness of partially demethylated intermediates lies in between. This has been shown by appropriate measurements both with artificial and natural membranes. We therefore conclude that the temporal sequence of steps nature has chosen for the sterol pathway evolved in time in response to selection pressures. Improvement of function was the driving force.

KONRAD BLOCH AFTER RETIREMENT The last experimentally based papers from Bloch’s lab were published in the early 1980s. He closed his lab at this time. He did not have to write any more grant applications! He had an office at Harvard and routinely went there each day. His scholarly interests continued during what he referred to as his permanent sabbatical (28). Writing an autobiography did not appeal to him. Instead he wrote a series of essays on topics he had tucked in the back of his mind (28). This collection of articles was published in 1994 by Yale University Press in a book titled Blondes in Venetian Paintings: The Nine-Banded Armadillo and Other Essays in Biochemistry. During a 1993 interview (1) he said, “I was struck by the fact that many women painted notably by Titian, Tintoretto, and Veronese, obviously from the upper classes, had blonde hair. If you meet a blonde in northern Italy, she is most likely a tourist from Scandinavia

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or Germany, or a peroxide blonde. It struck me that in the same paintings, the hair of the males is invariably very dark, in fact black. I put all these clues together, and concluded that the ladies in question must have known about bleaching hair centuries before hydrogen peroxide was discovered in 1812.” Further research led to the first essay in his book (28). Konrad Bloch died on October 15, 2000, at the age of 88. His contributions as a scientist, scholar, and a human being were enormous. He was one of the leading scientific figures of the 20th century. Moreover, as the accompanying articles in this volume testify, he was also a beloved mentor, teacher, colleague, and friend. REFERENCES 1. Konrad Bloch, interview by James J. Bohning at Harvard University, 22 March 1993 (Philadelphia: Chemical Heritage Foundation Oral History Transcript 109). 2. Bloch, K. (1987) Summing up. Annu. Rev. Biochem. 56, 1–19. 3. Bloch, K., and Clarke, H. T. (1938) N-Methylcysteine and derivatives. J. Biol. Chem. 125, 275–287. 4. Bloch, K. (1982) The structure of cholesterol and of the bile acids. Trends Biochem. Sci. 7, 334 –336. 5. Sonderhoff, R., and Thomas, H. (1937) Enzymic dehydrogenation of trideuteroacetic acid. Liebeg’s Ann. Chem. 530, 195–213. 6. Bloch, K., and Rittenberg, D. (1942) On the utilization of acetic acid for cholesterol formation. J. Biol. Chem. 145, 625– 636. 7. Bloch, K., and Rittenberg, D. (1944) The utilization of acetic acid for fatty acid synthesis. J. Biol. Chem. 154, 311–312. 8. Cornforth, J. W., Hunter, G. D., and Popja´ k, G. (1953) Studies of cholesterol biosynthesis. 1. A new chemical degradation of cholesterol. 2. Distribution of acetate carbon in the ring structure. Biochem. J. 54, 590 –597 and 597– 601. 9. Langdon, R. G., and Bloch, K. (1952) Biosynthesis of squalene and cholesterol. J. Am. Chem. Soc. 74, 1869. 10. Woodward, R. B., and Bloch, K. (1953) The cyclization of squalene in cholesterol synthesis. J. Am. Chem. Soc. 75, 2023– 2024. 11. Maudgal, R. K., Tchen, T. T., and Bloch, K. (1958) 1,2-Methyl shifts in the cyclization of squalene to cholesterol. J. Am. Chem. Soc. 80, 2589. 12. Cornforth, J. W., Cornforth, R. H., Pelter, M. G., Horning, G., and Popja´ k, G. (1959) Rearrangement of methyl groups in the enzymic cyclization of squalene to lanosterol. Tetrahedron 5, 311–339. 13. Wolf, D. E., Hofmann, C. H., Aldrich, P. E., Skeggs, H. R.,

Wright, L. D., and Folkers, K. (1956) ␤-Hydroxy-␤-methyl-␦valerolactone (divalonic acid), a new biological factor. J. Am. Chem. Soc. 78, 4499. 14. Tavormina, P. A., Gibbs, M. H., and Huff, J. W. (1956) The utilization of ␤-hydroxy-␤-methyl-␦-valerolactone in cholesterol biosynthesis. J. Am. Chem. Soc. 78, 4498 – 4499. 15. Bloch, K. (1965) The biological synthesis of cholesterol. Science 150, 19 –28. 16. Bloomfield, D. K., and Bloch, K. (1960) The formation of delta9-unsaturated fatty acids. J. Biol. Chem. 236, 337–345. 17. Schroepfer, G. J., and Bloch, K. (1963) Enzymatic stereospecificity in the dehydrogenation of stearic acid to oleic acid. J. Am. Chem. Soc. 85, 3310. 18. Bloch, K. (1969) Enzymatic synthesis of mono-unsaturated fatty acids. Acc. Chem. Res. 2, 193–202. 19. Lennarz, W. J., Light, R. J., and Bloch, K. (1962) A fatty acid synthetase from E. coli. Proc. Natl. Acad. Sci. USA 48, 840 – 846. 20. Goldman, P., Alberts, A. W., and Vagelos, P. R. (1961) Requirement for a malonyl-CoA–CO 2 exchange reaction in long-chain but not short-chain fatty acid synthesis in Clostridium kluyveri. Biochem. Biophys. Res. Commun. 5, 280 –285. 20a.White, H. (2002) Konrad Bloch, evolution, and the RNA world. Biochem. Biophys. Res. Commun. 292, 1267–1271. 21. Bloch, K., and Vance, D. E. (1977) Control mechanisms in the synthesis of saturated fatty acids. Annu. Rev. Biochem. 46, 263– 298. 22. Brindley, D. N., Matsmura, S., and Bloch, K. (1969) Mycobacterium phlei fatty acid synthetase—A bacterial multienzyme complex. Nature 224, 666 – 669. 23. Ilton, M., Jevans, A. W., McCarthy, E. D., Vance, D., White, H. B., and Bloch, K. (1971) Fatty acid synthetase activity in Mycobacteria phlei: Regulation by polysaccharides. Proc. Natl. Acad. Sci. USA 68, 87–91. 24. Vance, D. E., Mitsuhashi, O., and Bloch, K. (1973) Purification and properties of the fatty acid synthetase from Mycobacterium phlei. J. Biol. Chem. 248, 2303–2309. 25. Knoche, H., Esders, T. W., Koths, K., and Bloch, K. (1973) Palmityl-CoA inhibition of fatty acid synthesis—Relief by bovine serum albumin and mycobacterial polysaccharides. J. Biol. Chem. 248, 2317–2322. 26. Bloch, K. (1976) On the evolution of a biosynthetic pathway in reflections on biochemistry (Kornberg, A., Horecker, B. L., Cornudella, L., and Oro, J., Eds.), pp. 143–150, Pergamon Press, Oxford. 27. Bloch, K. (1983) Sterol structure and membrane function. CRC Crit. Rev. Biochem. 14, 47–92. 28. Bloch, K. (1994) Blondes in Venetian Paintings, the NineBanded Armadillo, and Other Essays in Biochemistry, Yale Univ. Press, New Haven, CT.

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