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Biographical Article

Esmond Emerson Snell (1914–2003)

Scientist Emeritus, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0830 and ^* Distinguished Professor Emeritus, Department of Biochemistry and Biophysics, Iowa State University, Ames, IA 50011-3260

¹To whom correspondence should be addressed. E-mail: EdithM{at}intra.niddk.nih.gov.

Esmond Emerson Snell (1914–2003) Photograph reprinted with permission from the Annual Review of Biochemistry

Esmond Snell was a pioneer and a giant in nutritional and biochemical research. His early work on the nutritional requirements of lactic acid bacteria resulted in the development of the microbiological assay (Table 1) and led to the discovery and analysis of a number of vitamins, antivitamins, and growth factors (Table 2) (1,2). His discovery of two new forms of vitamin B-6, pyridoxal and pyridoxamine, led to an understanding of the chemical reactions of vitamin B-6 and the catalytic mechanism of B-6–dependent enzymes. He was a Professor of Biochemistry at the University of Wisconsin (1945–1951), Professor of Chemistry at the University of Texas (1951–1956 and 1976–1990) and Professor of Biochemistry at the University of California, Berkeley (1956–1976). During his long and productive career, he trained over 30 Ph.D. students, had more than 40 postdoctoral fellows and senior associates, and published 400 scientific papers and reviews, which were always written accurately, clearly, and carefully. He received numerous awards, including the Meade-Johnson Vitamin B Complex Award in 1946, the Osborne-Mendel Award in 1951 from the American Institute of Nutrition, and the William C. Rose Award in 1985 from the American Society of Biological Chemists. He was elected to the National Academy of Sciences in 1955 and was elected a Fellow of the American Institute of Nutrition in 1982. He served on a number of government committees and editorial boards. He was the editor of the Annual Review of Biochemistry from 1968 to 1983.

Development and use of microbiological assays

Esmond started his graduate work at the University of Wisconsin in Madison in 1935 doing research in the laboratory of Professor W. H. Peterson, a biochemist who was interested in microbial metabolism. Esmond’s thesis project was to identify growth factors for lactic acid bacteria, which require complex media for growth. The experimental approach was to start with a simple medium and to determine what supplements were necessary for growth. The only B vitamins known at this time were riboflavin and thiamin. Esmond’s first publication with F. M. Strong in 1937 reported that a potato extract was the source of an unknown growth factor. Much later, using the microbiological assay provided for this growth factor (Table 1), Lester Reed and his co-workers isolated crystalline lipoic acid, which proved to be an essential cofactor for pyruvate dehydrogenase. Esmond’s later work resulted in the discovery of several vitamins, growth factors, and inhibitors (Table 2) and in the development of the microbiological assay for following the purification of these substances and for determining their concentrations in nature (Table 1). He published in 1939 an assay for riboflavin, which was the first widely used microbiological assay method for a vitamin; this assay served as a prototype for assays for each of the B vitamins. The method gave results comparable to those obtained by a much lengthier, cumbersome, and expensive rat assay. While at Madison, Esmond also published microbiological assays for pantothenic acid and nicotinic acid (Table 1). Once all of the vitamins required by the lactic acid bacteria were identified and commercially available, the vitamins could be added to the basal medium along with a complete assortment of amino acids. Individual amino acids required for growth could be determined by single omissions; organisms that required an amino acid could be used for quantitative assay of that amino acid (Table 1). These assays were widely used until the automatic ion exchange procedures were developed; microbiological assays are still used for some purposes today. The use of microbiological assays instead of animal assays for vitamins and amino acids resulted in untold savings of time and money.

Esmond left Madison in 1939 for his first job as a postdoctoral research associate with Roger J. Williams at the University of Texas in Austin. He became Assistant Professor in 1941. At the University of Texas he developed a microbiological assay for the recently discovered vitamin, biotin. He used this assay for the first purification and characterization of avidin, a protein in egg white that binds biotin very tightly. Next, using Streptococcus lactis R. as a test organism, Esmond Snell with Hershel Mitchell and Roger Williams purified from 4 tons of spinach a growth factor that they named "folic acid" (Table 2). The report of this work (3) was termed "A Nutrition Classic." This microbiological assay is still used for the determination of folates in the blood.

In the course of investigations of microbiological assays for pyridoxine with different microorganisms, Esmond discovered 2 new forms of vitamin B-6. He observed that an extraordinarily high amount of pyridoxine was required for the growth of S. faecalis when filter-sterilized pyridoxine was added to the medium, but that much lower amounts were required when pyridoxine was heat-sterilized with the medium. The growth effect of pyridoxine for the yeast, Saccharomyces carlsbergensis, was the same under both conditions. The finding that pyridoxine also became much more active for S. faecalis after ammonia treatment or mild oxidation suggested that pyridoxine was converted to aldehyde and amine forms. The structures of these compounds were determined by synthesis in collaboration with Karl Folkers and his group at Merck & Co. and were named pyridoxal and pyridoxamine, respectively (Table 2). Esmond then developed differential assays for the 3 forms of vitamin B-6 in natural materials using 3 microorganisms that had different nutritional requirements for the forms (Table 1). After returning to the University of Wisconsin in 1945, he and his student, Jesse Rabinowitz, discovered pyridoxamine phosphate.

Esmond’s research at Madison also resulted in the discovery that D-alanine is a growth factor for S. faecalis in the absence of vitamin B-6 and that bacterial cell walls contain most of the cellular D-alanine. He also discovered that a growth factor for Lactobaccilus bulgaricus was an amide of cysteamine with panthothenic acid. Gene Brown’s Ph.D. thesis was on this subject. Gene recalls "I remember vividly the Saturday morning when Es and I got together to decide what to call this substance which we had been calling LBF (or the Lactobacillus bulgaricus Factor) before we identified it. I was pleased that Es decided on pantetheine, a name I had submitted" (Table 2). Esmond and his colleagues also found that putrescine, spermine, and spermidine were growth factors for Hemophilus parainfluenzae and clarified the roles of peptides as growth factors for lactic acid bacteria. They established that peptides of a given amino acid may be more active than the free amino acid if the cell is unable to transport the amino acid, if the free amino acid is readily degraded, or if the uptake of the amino acid is blocked by antagonistic amino acids. Esmond’s group also carried out important studies on pantothenate degradation and synthesis and on the pathway of vitamin B-6 degradation by bacteria [reviewed in (2)].

Mechanism of catalysis by vitamin B-6

While investigating the natural forms of vitamin B-6, Esmond found that pyridoxal and pyridoxamine were readily interconverted by a fully reversible nonenzymatic transamination reaction with glutamate and -ketoglutarate (Eq. 1). The reaction also occurred with other -amino acids and -ketoacids, including aspartate and oxaloacetate (Eq. 2). Coupling of two such reactions (e.g., Eqs. 1 and 2) would give a fully reversible nonenzymatic transamination reaction (Eq. 3) in which pyridoxal and pyridoxamine would act only as catalysts. Esmond proposed in 1944 that these compounds might play a similar role in enzymatic transamination.

(1)

(2)

(3)

Esmond and his colleagues next undertook a detailed investigation of the mode of action of vitamin B-6. Esmond was especially interested in the nonenzymatic transamination reactions. In 1949 he and his student David Metzler initiated a detailed study. David recalls, "One day Esmond walked into the laboratory with a bottle of a new compound Versene (EDTA) and asked what effect it would have on the reaction. EDTA was strongly inhibitory, a finding that led to the discovery that the reaction was metal ion–dependent and established conditions for obtaining reproducible kinetic results." Glutamate and alanine underwent rapid transamination in a buffered Al³⁺-catalyzed reaction. However, serine was converted to pyruvate, and threonine was cleaved rapidly to acetaldehyde and glycine. These and other reactions were recognized as simulating closely the corresponding reactions catalyzed in living organisms by pyridoxal phosphate –dependent reactions [reviewed in (4)]. Experiments with analogs and derivatives of pyridoxal, several of which were synthesized by Myoshi Ikawa, established that the carbonyl, hydroxyl, and ring nitrogen of pyridoxal were necessary for rapid reaction. These studies led to the proposal in 1954 of a general mechanism for the action of vitamin B-6–dependent enzymes (5). This mechanism, which was similar to one proposed independently by A. E. Braunstein and M. M. Shemyakin, helped to explain the multiple roles played by vitamin B-6 in living organisms. Pyridoxal phosphate–dependent enzymes catalyze a wide variety of reactions including transamination, ß-elimination, ß-replacement, and decarboxylation, racemization, and aldol cleavage reactions. Subsequent investigators of these important pyridoxal phosphate–dependent enzymes regard Esmond Snell and Alexander Braunstein as the fathers of vitamin B-6.

Catalytic mechanism of B-6–dependent enzymes and pyruvoyl enzymes

Esmond next turned to studies of vitamin B-6–dependent enzymes to test some aspects of the proposed general mechanism. The characterization of a pyridoxamine-pyruvate transaminase in 1962 demonstrated that the phosphate group of pyridoxal phosphate is not essential for enzymatic catalysis. Edith Wilson Miles recalls "My Ph.D. studies published in 1962 demonstrated the enzymatic cleavage of -methylserine to D-alanine and formaldehyde and showed that this reaction does not require labilization of an -H." The results of extensive studies of tryptophanase supported the proposal that ß-elimination and ß-replacement reactions proceed through an -aminoacrylate intermediate [reviewed in (6)]. Esmond investigated several other pyridoxal phosphate–dependent enzymes including D-serine dehydratase, arginine decarboxylase, and histidine decarboxylase.

Interestingly, there are two types of histidine decarboxylase. One type, which is pyridoxal phosphate–dependent, is found in gram-negative bacteria and in mammals. The second type, discovered by Esmond, is found in gram-positive organisms (e.g., Lactobacillus 30a) and contains an essential, covalently bound pyruvoyl prosthetic group that participates as a Schiff base in the catalysis of decarboxylation [reviewed in (7,8)]. Esmond and colleagues proved that the pyruvoyl group arises from a specific serine residue in the proenzyme by a previously unobserved, unique, intrachain, nonhydrolytic cleavage reaction. Peptide chain cleavage is coupled to an ß-elimination reaction to form active enzyme that contains 2 chains, one of which has an N-terminal pyruvoyl residue.

Several other enzymes were found subsequently to have pyruvoyl coenzymes, i.e., S-adenosylmethionine decarboxylase, L-aspartate--decarboxylase, phosphatidylserine decarboxylase, and proline and glycine reductases. The cloning and determination of the nucleotide sequence of the pyruvoyl-dependent histidine decarboxylase from Lactobacillus 30a by J. D. Robertus and his colleagues in 1986 made it possible for D. T. Gallagher and M. L. Hackert to determine the 3-dimensional structure of this enzyme in 1989 (9). The structure permitted identification of the active site and analysis of the structural basis of catalysis. The postulated roles of various residues are consistent with the results of site-directed mutagenesis studies by the Snell and Robertus groups.

Esmond’s roles as a mentor and his philosophy of science

During his work at the University of Texas in the early 1940s, Esmond worked actively in the laboratory, often alone. When he moved to the University of Wisconsin in Madison in 1945 and had many more students and heavier teaching responsibilities, Esmond found that he needed to spend most of his time in his office. His students at Madison included J. C. Rabinowitz, J. T. Holden, E. J. Herbst, L. M. Henderson, H. P. Broquist, D. E. Metzler, and G. M. Brown. David Metzler remembers that "everybody was expected to be behind the bench at 8 a.m. six days a week. Sitting at a desk didn’t count as real work. Esmond was very well read and could point us in new directions. We were always amazed at how many hours he spent in his office behind closed doors writing. He only came out for quick daily tours around the lab. We also saw him on a rigid weekly schedule in his office. He expected not only a properly kept notebook but also manuscripts of reports for publication, often in the Journal of the American Chemical Society or the Journal of Biological Chemistry. These manuscripts were edited unmercifully until they were short and to the point but he always put us as first authors. Snell was an excellent writing teacher. He treated us all with respect, even when work was not going well. We all liked Es." Gene Brown remembers "Esmond took me under his wing and taught me how to be a professional scientist. Without question, he has been the one person who has made the most impact on me and my life as a person and a scientist. He and Mary became my surrogate parents during my years as a graduate student."

Esmond acknowledged 2 longer-term associates who were especially helpful in assuring continuity of projects and in maintaining a workable laboratory (2). Hayato Kihara filled this function during the early years through 1960. David Metzler recalls that in Wisconsin "Hy ordered chemicals and glassware and insisted that we keep our workplaces clean and orderly." He also published with Esmond an important series of 10 papers on peptides and bacterial growth. Beverly Guirard was one of Esmond’s early associates at the University of Texas in the early 1940s who participated in the identification of the natural forms of vitamin B-6. Beverly joined Esmond’s group when he returned to the University of Texas in 1951 and remained active in research in the group through the years in Berkeley (1956–1976) and the return to Austin in 1976 until her retirement in the 1990s. Her important work included studies of histidine decarboxylase from Lactobacillus 30A and the pyridoxal phosphate–dependent histidine decarboxylase from Morganella AM-15. During Esmond’s early days in Berkeley, his laboratory was very busy with several members who had come from the University of Texas, new graduate students (C. T. Goodhue, E. M. Wilson, and W. A. Newton) and postdoctoral fellows and senior associates (H. Kihara, M. Ikawa, G. Kalyankar, A. N. Radhakrishnan, D. B. McCormick, J. Mora, T. K. Sundaram, W. B. Dempsey, B. M. Guirard, and others). Edith Wilson Miles recalls: "On Saturday mornings Beverly prepared French roast coffee in the style of her native Louisiana and Esmond would come out of his office to join those who were working; Saturday work was no longer required but was still appreciated." Donald McCormick recalls: "Lunch breaks in Strawberry Canyon where the Snell group could enjoy a bit of nature in the late 50s and early 60s." Esmond also influenced many graduate students through his excellent lectures at the University of Wisconsin, the University of Texas, and the University of California.

The arrival of Hiroshi Wada in 1961 marked the beginning of important contributions by Japanese scientists to the laboratory. He was followed by a series of other Japanese scientists including Y. Morino, H. Kagamiyama, and H. Hayashi. All of these later became Professors in Japan. Esmond had a sabbatical stay at Osaka University in 1971. One day, after his lecture, a student asked him to write on a fancy paperboard a message for the students. He wrote "Hard work on interesting problems is enjoyable and preferable to aimless wasting of leisure time. It may also lead to unexpected findings that give insights into important related problems. Such unexpected findings—sometimes called "luck"—frequently happen to the active researcher, but only rarely to those who prefer talk to study and work. So one should study and work hard, on interesting problems of any nature, with the purpose of explaining nature and helping others." Many Japanese scientists have copies of this statement, which summarizes Esmond’s philosophy of science.

The last 60 years have seen tremendous progress in studies of nonenzymatic and enzymatic B-6 catalysis using modern techniques of protein purification, recombinant genetics, and structural biology. Much of this progress has been reported in a series of International Symposia on Pyridoxal Catalysis, which later included other Carbonyl Compounds as Cofactors, in Rome (1962), Moscow (1966), Nagoya (1967), Leningrad (1974), Toronto (1979), Athens (1983), Turku (1987), Osaka (1990), Capri (1994), Santa Fe (1999), and Southampton (2002). The meeting in Turku memorialized the death of Alexander E. Braunstein (1902–1986). The meeting in Santa Fe presented a special honor to Esmond Snell. Esmond retired from his laboratory at the University of Texas in 1990. He and Mary enjoyed many interesting travels in their later years as well as contacts with family and former colleagues.

	ACKNOWLEDGMENTS

 
The authors gratefully acknowledge the contributions of materials and comments from L. Reed, M. L. Hackert, D. B. McCormick, B. M. Guirard, G. M. Brown, J. F. Kirsch, H. Tabor, H. Hayashi, and R. L. Sanders. We acknowledge that the photograph of Esmond E. Snell is reprinted, with permission, from the Annual Review of Biochemistry, Volume 62 © 1993 by Annual Reviews www.annualreviews.org.

Manuscript received 28 June 2004.

	LITERATURE CITED

TOP LITERATURE CITED

 

1. Snell, E. E. (1989) Nutrition research with lactic acid bacteria: a retrospective view. Annu. Rev. Nutr. 9:1-19.[Medline]

2. Snell, E. E. (1993) From bacterial nutrition to enzyme structure: a personal odyssey. Annu. Rev. Biochem. 62:1-27.[Medline]

3. Mitchell, H. K., Snell, E. E. & Williams, R. J. (1941) The concentration of "folic acid.". J. Am. Chem. Soc. 63:2284-2285.

4. Snell, E. E. (1958) Chemical structure in relation to biological activities of vitamin B6. Vitam. Horm. 16:77-125.[Medline]

5. Metzler, D. E., Ikawa, M. & Snell, E. E. (1954) A general mechanism for vitamin-B6 catalyzed reactions. J. Am. Chem. Soc. 76:648-652.

6. Snell, E. E. (1975) Tryptophanase: structure, catalytic activities and mechanism of action. Adv. Enzymol. 42:389-446.

7. Recsei, P. A. & Snell, E. E. (1984) Pyruvoyl enzymes. Annu. Rev. Biochem. 53:357-387.[Medline]

8. van Poelje, P. D. & Snell, E. E. (1990) Pyruvoyl-dependent enzymes. Annu. Rev. Biochem. 59:29-59.[Medline]

9. Gallagher, T., Snell, E. E. & Hackert, M. L. (1989) Pyruvoyl-dependent histidine decarboxylase. Active site structure and mechanistic analysis. J. Biol. Chem. 264:12737-12743.[Abstract/Free Full Text]

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