Portal de Periódicos da CAPES

Using Studies on Tryptophan Metabolism to Answer Basic Biological Questions

2003; Elsevier BV; Volume: 278; Issue: 13 Linguagem: Inglês

10.1074/jbc.x200012200

1083-351X

Charles Yanofsky,

Enzyme Structure and Function

In my youth I was overwhelmed by the variety of forms of life around me. Yes, while growing up in New York City! As a student at the Bronx High School of Science my teachers made every effort to convince me that no pursuit could be more exciting or rewarding than searching for explanations for the basic processes common to life. I agreed, but I knew this decision was insufficient, for I would have to choose the area of science that was just right for me. I was aware that major unanswered questions existed in all fields of science, particularly regarding the relationship of biochemistry to genetics, the two subjects that interested me most as a high school student. I decided to major in biochemistry, and enrolled at the City College of New York. I completed a year and a half of college study before being drafted into the army in the spring of 1944. I served in the infantry as a cannoneer during World War II. I fought in the Ardennes in the Battle of the Bulge. Understandably this was an awesome experience. Upon returning to college after the war I was more determined than ever to pursue a career in research. When faced with selecting a Ph.D. program to apply to, I received excellent advice from a knowledgeable professor and textbook author, Benjamin Harrow, chairman of the Biochemistry Department at City College of New York. He suggested exploring gene-enzyme relationships with Neurospora crassa as the ideal project for me. I agreed and applied to do my graduate work with George Beadle at Caltech or Edward Tatum at Yale. I was rejected by Caltech but fortunately was accepted by Yale. As it turned out, my mentor in graduate school at Yale was not Edward Tatum; it was David Bonner. Bonner had moved with Tatum from Stanford to Yale and had become his research associate. During the year I applied for admission to Yale, Tatum decided to return to Stanford. Fortunately for me, Bonner stayed on at Yale and took over direction of Tatum's remaining group. Bonner, a wonderful advisor, believed it was in the best interests of both student and advisor to have each student work independently on a well defined project. If successful, he said, we would receive partial credit for our discoveries and would qualify for a faculty position. For most beginning graduate students, selecting a project and deciding how to proceed is relegated to your research mentor and would reflect his or her research preferences. By choosing a specific scientist as your advisor you recognize the importance of his or her contributions. In my initial meeting with Bonner at Yale in June of 1948, as I recall, he handed me a fuzzy culture of a niacin-requiring mutant of Neurospora and gave me advice on how to go about identifying the niacin pathway intermediate this mutant was presumed to accumulate. Our ultimate goal, he said, was identifying all the intermediates in the niacin pathway so this knowledge could be exploited in investigations on gene-enzyme relationships. I was the only laboratory member assigned this type of project, probably because Bonner was aware that my background was principally in biochemistry. This project captured my full attention, and fortunately, I was successful. We identified two intermediates accumulated by niacin-requiring mutants, quinolinic acid and a derivative of kynurenine. The knowledge I acquired in these studies served as a valuable resource in decision making throughout the early stages of my career. Upon reviewing my research accomplishments and considering what I might emphasize in this article, I was most impressed by the variety of basic biological questions the members of my group have addressed. Early in my career I decided that one of my primary research objectives would be to provide a thorough understanding of all aspects of tryptophan metabolism and to use this knowledge in explaining basic processes of biology. In fact, tryptophan metabolism was the focus of most of my research. However, during the early stages of my career I did not appreciate the variety of scientific questions that I would have the opportunity to address using tryptophan metabolism as my experimental system. Our studies contributed to knowledge on the niacin and tryptophan biosynthetic pathways, enzyme structure/function relationships, organization of genes and operons, the existence of gene-protein colinearity, the molecular basis of suppression, coupling of transcription with translation, regulation of transcription, how tryptophan and tryptophan-tRNA serve as regulatory signals, and the regulatory mechanisms microorganisms use to control tryptophan synthesis and its degradation. The unanticipated role of RNA in regulation, transcription attenuation, was and continues to be one of our major interests. We had no inkling until the 1990s, when bacterial genomes were beginning to be sequenced, that attenuation was so widely used in nature. While we were conducting our investigations on tryptophan metabolism evolutionary questions continually arose. As soon as we understood the features of tryptophan metabolism in one organism we wished to know whether other organisms use the same genes, reactions, and regulatory processes. Despite my personal commitment to tryptophan metabolism, in the early 1980s I returned to studies withN. crassa as an experimental organism, addressing other important questions. The lesson to be learned from my experiences, I believe, is to always be on the alert. Important unanswered questions you never anticipated will invariably arise from the results of your current research. It may develop that your chosen experimental system is ideal for answering these questions. Throughout this article I will describe examples taken from my career, where answers led to questions I felt we should address. When I arrived at Yale 1n 1948 most members of the Bonner group were coping with the most significant question then concerning the Neurospora scientific community: how to establish the nature of the gene-enzyme relationship. It was some years after Beadle and Tatum (1Beadle G.W. Tatum E.L. Genetic control of biochemical reactions in Neurospora..Proc. Natl. Acad. Sci. U. S. A. 1941; 27: 499-506Google Scholar) had first proposed the one gene, one enzyme, one biochemical reaction hypothesis. Following the pioneering studies of Garrod in the early 1900s, linking heredity with metabolism, there were numerous observations relating metabolic defects with genetic disorders. Beadle and Tatum cemented this relationship in the early 1940s by selecting an organism, N. crassa, that could be used to isolate nutritional mutants. These mutants could then be genetically characterized to establish whether their inability to carry out specific biochemical reactions was because of mutations in specific genes. Most importantly, they observed that there was a one to one relationship between gene and biochemical reaction. Despite these findings, when I was completing my graduate studies in 1951 most scientists were skeptical of the validity of the one gene-one enzyme concept. At this time very little was known about the molecular nature and structure of genetic material or the structure of proteins, and virtually nothing was known about protein synthesis. It was not until the early 1950s that the findings of Hershey and Chase (2Hershey A.D. Chase M. Independent functions of viral protein and nucleic acids in growth of bacteriophage..J. Gen. Physiol. 1952; 36: 39-56Google Scholar) and an earlier finding by Avery et al. (3Avery O.T. MacLeod C.M. McCarty M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types..J. Exp. Med. 1944; 79: 137-157Google Scholar) convinced most of us that genetic material was most likely DNA, and it was not until 1953 that Jim Watson and Francis Crick (4Watson J.D. Crick F.H.C. Molecular structure of nucleic acids..Nature. 1953; 171: 737-738Google Scholar) described their elegant structure for DNA. Following these major contributions we accepted as proven that the genetic material of most organisms was double-stranded DNA. Furthermore, it was not until the late 1950s that Seymour Benzer's (5Benzer S. The elementary units of heredity. In The Chemical Basis of Heredity. Johns Hopkins University Press, Baltimore, MD1957: 70-93Google Scholar) fine structure genetic analyses with the rII locus of phage T4 equated the genetic map with the structure of DNA. Similarly, it was not until the early 1950s that Sanger's studies (6Sanger F. The arrangement of amino acids in proteins..Adv. Protein Chem. 1952; 7: 1-28Google Scholar) with insulin established that proteins consist of linear sequences of amino acids. While I was in graduate school the goal considered most important by members of the Beadle-Tatum school was to identify a specific enzymatic reaction for which defective mutants could be isolated and then determine whether these mutants lacked that enzymatic activity. Our hope was that studies like these would provide definitive proof for the one gene-one enzyme hypothesis. Several members of the Bonner group were following this approach. Naomi Franklin, Otto Landman, Gabriel Lester, and Howard Rickenberg were examining one of the most popular experimental enzymes during this period, β-galactosidase, from bothNeurospora and Escherichia coli. They were hoping to use the knowledge and techniques being provided by Monod, and subsequently by Jacob and Monod and their exceptional coworkers, to explore the Beadle-Tatum gene-enzyme concept more directly. Impressed by this overriding goal of my mentor and the determination of my fellow students, I decided that I too should follow this path. In my third and last year of graduate study, 1950–1951, I abandoned my niacin pathway studies and initiated a search for the ideal "gene-enzyme" experimental system. No one in our group at Yale was contemplating what today would be considered the most obvious experimental approach: isolating and sequencing a specific gene and comparing this sequence with the amino acid sequence of its polypeptide product. Neither genes nor proteins could be analyzed in this way; we did not yet know that genetic material was DNA or that proteins consisted of linear sequences of amino acids. At this time the prevailing view in the field of genetics was that chromosomes consist of linear arrays of genes arranged like "beads on a string." It was assumed that each gene was indivisible by genetic recombination. If these views were correct how could we determine the relative positions of independent mutational changes in a specific gene, except by structural analysis, which was not possible? We decided that our next step on the gene-enzyme problem should be to demonstrate convincingly that all mutants altered at a single genetic locus lack the specific enzyme that catalyzes the corresponding reaction. By the late 1940s numerous nutritional mutants of N. crassahad been isolated, many requiring the same metabolite. It was evident that amino acids, vitamins, purines, and pyrimidines are all synthesized by sequential enzyme-catalyzed reactions, mostly in separate pathways. However, these pathways were just beginning to be defined. Genetic analyses with these mutants established a very impressive one-to-one relationship between altered gene and loss of a specific biochemical reaction; this was the experimental basis of the Beadle/Tatum concept. It was also evident that a unique set of genes was associated with each metabolic pathway. However, very few of the enzymes in each newly discovered pathway had been identified, and those that were known did not catalyze reactions that were defective in the nutritional mutants that had been isolated. One of the earliest opportunities to examine mutants lacking a specific enzyme was provided by the findings of Umbreit et al. in 1946 (7Umbreit W.W. Wood W.A. Gunsalus I.C. The activity of pyridoxal phosphate in tryptophan formation by cell-free enzyme preparations..J. Biol. Chem. 1946; 165: 731-732Google Scholar). They demonstrated that extracts of wild type Neurospora contain an enzyme they named tryptophan desmolase, which catalyzes the last reaction in tryptophan synthesis, the covalent joining of indole withl-serine to form l-tryptophan. Tryptophan-requiring mutants of Neurospora had been identified that could not grow on indole; therefore these mutants should lack this enzyme activity if the Beadle/Tatum hypothesis were correct. Joseph Lein and Dave Hogness, of Hershell Mitchell's laboratory at Caltech, examined extracts of one such mutant, namedtd1, and reported that yes, it did lack tryptophan desmolase activity (8Mitchell H.K. Lein J. A Neurospora mutant deficient in the enzymatic synthesis of tryptophan..J. Biol. Chem. 1948; 175: 481-482Google Scholar, 9Hogness D.S. Mitchell H.K. Genetic factors influencing the activity of tryptophan desmolase in Neurospora crassa..J. Gen. Microbiol. 1954; 11: 401-411Google Scholar). Having spent my first 2 years studying niacin and tryptophan metabolism in Neurospora, I decided that tryptophan desmolase was promising as a potential subject for gene-enzyme analyses. I initiated my studies by partially purifying and further characterizing the wild type enzyme and confirming the absence of tryptophan desmolase activity in extracts of mutant td1. I also examined a second mutant altered at the same locus, mutant td2, and showed that it too lacked tryptophan desmolase activity (10Yanofsky C. The effects of gene change on tryptophan desmolase formation..Proc. Natl. Acad. Sci. U. S. A. 1952; 38: 215-226Google Scholar). Excited by the simplicity of this enzyme assay and these positive results, members of the Bonner group turned to isolating 20 additional mutants defective in the conversion of indole to tryptophan. We showed that each was genetically altered at the td locus and each lacked tryptophan desmolase activity. These initial findings were very encouraging, and they supported the basic assumption of the Beadle/Tatum concept. In the course of my studies with mutant td2 one culture grew in media lacking tryptophan. Instead of discarding this culture, we analyzed it genetically and discovered that its ability to grow without tryptophan was due to an unlinked suppressor mutation. The properties of this suppressed td mutant raised a new, then unanswerable, question. How does a suppressor mutation, a mutation in a gene other than the td gene, restore growth without tryptophan? My enzyme analyses revealed that the suppressor mutation acted by restoring the organism's ability to form an active tryptophan desmolase (10Yanofsky C. The effects of gene change on tryptophan desmolase formation..Proc. Natl. Acad. Sci. U. S. A. 1952; 38: 215-226Google Scholar). Probing still further, I observed that thetd2 suppressor gene was allele-specific; it had no effect on mutant td1. Obviously, then, mutants td1 andtd2 must have different alterations at the tdlocus. We next performed "reversion" analyses with all ourtd mutants and isolated several additional suppressors. Most of these restored tryptophan desmolase activity only when combined with their respective td mutant allele. On the basis of these findings we rephrased our previous question, as follows. If there is a one-to-one relationship between gene and enzyme and only td mutants lack tryptophan desmolase activity, how does a mutation in a gene distinct from the td locus restore this enzyme activity? My thoughts on possible explanations temporarily diverted attention from my primary objective, establishing the basis of the one gene-one enzyme relationship. I considered our suppression findings to be extremely interesting and believed that their explanation might provide additional insight into this relationship. This experience, I believe, was largely responsible for many of my subsequent decisions on how to proceed in planning future research. I decided then that our knowledge of basic biological processes was so poor it would be foolish to ignore interesting unexplained observations. Following this line of reasoning I set out to compare the properties of tryptophan desmolase isolated from the wild type strain and from several suppressed mutants. Throughout this period we were frustrated at how little we could do experimentally. The existing molecular technology was clearly inadequate. With a close friend and former member of the Bonner group, Sigmund Suskind, then a postdoctoral fellow performing immunological research at another institution, we designed a different approach that we thought might provide additional insight into the gene-enzyme relationship, The question we set out to answer was the following.Does suppressible mutant td2, but not non-suppressible mutant td1, produce an inactive form of the tryptophan desmolase enzyme?Using my partially purified wild type enzyme as antigen, Suskind prepared a rabbit antiserum that inhibited wild type tryptophan desmolase activity. We used this antiserum in a successful weekend experiment at Yale, analyzing extracts of mutants td1 andtd2 for an inactive tryptophan desmolase-like protein that would cross-react with our antiserum (11Suskind S.R. Yanofsky C. Bonner D.M. Allelic strains of Neurospora lacking tryptophan synthetase: a preliminary immunochemical characterization..Proc. Natl. Acad. Sci. U. S. A. 1955; 41: 577-582Google Scholar). Mutant td2extracts did in fact contain such a cross-reacting material, for which we coined the term "CRM," whereas extracts of mutant td1did not. Comparable analyses were then performed with extracts of our other td mutants. All our suppressible mutants were shown to be CRM+, whereas all our non-suppressible mutants were CRM−. These findings implied, incorrectly, that suppression can restore a functional enzyme only if a mutant produces an inactive form of the wild type enzyme. (There are several reasonable explanations for our inability to isolate suppressors of our CRM− Neurospora mutants, which probably had chain termination mutations in the td gene.) On the basis of our findings I drew a number of interesting conclusions. I presented these at a very exciting symposium entitled "Enzymes, Units of Biological Structure and Function" held at the Henry Ford Hospital in Detroit in 1955 (Fig.1) (12Yanofsky C. Gene interactions in enzyme synthesis..in: Henry Ford Hospital International Symposium: Enzymes, Units of Biological Structure and Function. Academic Press Inc., New York1956: 147-160Google Scholar). My interpretations were of course influenced by new knowledge on DNA and protein structure and the mechanism of protein synthesis. I concluded that "the tdlocus is the only chromosomal area which directly controls tryptophan synthetase formation" (the accepted name had just been changed from desmolase to synthetase). I also concluded that "the tdlocus represents a physiologically indivisible unit, damage to any part of which results in a defect in tryptophan synthetase formation." I stated that "it would seem likely that different portions of thetd locus are concerned with the synthesis of different parts of the tryptophan synthetase molecule." In attempting to explain how a suppressor mutation restores enzyme activity I postulated that "some product of a suppressor gene cooperates with the altered template in the formation of small amounts of tryptophan synthetase." Looking back on these interpretations, they were all naive guesses, but they proved to be correct. Unfortunately the experimental tools and approaches needed to establish their molecular validity were not available. These studies on missense suppression preceded the enormous interest in suppression aroused by studies on the genetic code and on nonsense mutations. As is so often the case, the significance of a finding is not appreciated until additional relevant knowledge is acquired. At this stage in my career I was deeply committed to doing everything I could to provide additional insight into the gene-enzyme relationship. I was disappointed at the difficulty I was experiencing attempting to purify the tryptophan synthetase of Neurosporaand initiated a search for a more suitable experimental enzyme. My first thought was to identify an enzyme in the tryptophan to niacin pathway from E. coli or Bacillus subtilis, because these organisms were developing as more ideal experimental subjects for biochemical analyses. I performed radioisotope-labeling experiments with these two organisms, hoping to show that one or both synthesizes niacin from tryptophan. My findings provided a disappointing conclusion; neither organism synthesizes niacin from tryptophan (13Yanofsky C. The absence of a tryptophan-niacin relationship in Escherichia coli and Bacillus subtilis..J. Bacteriol. 1954; 68: 577-584Google Scholar). This negative result eliminated enzymes of the niacin pathway from my list of possibilities. While performing these studies I was offered a faculty position in the outstanding Microbiology Department at the Western Reserve University School of Medicine. I decided to accept their offer and left Yale for Cleveland in 1954. As a beginning Assistant Professor I felt it would be wiser to shift my research objectives to a well defined problem, one for which I could foresee obtaining definitive answers. I relied on my prior scientific experience and chose determining the missing reactions in the tryptophan biosynthetic pathway. Although many different classes of tryptophan auxotrophs had been isolated in Neurospora,E. coli, and other organisms, only two intermediates in the tryptophan pathway had been identified, anthranilate and indole. I chose an enzymological approach in attempting to identify the intermediates in the pathway and initiated my studies by analyzing extracts of wild type and different classes of tryptophan auxotrophs ofE. coli. My efforts focused on unidentified intermediates in the tryptophan biosynthetic pathway were successful. Using an enzymological approach we succeeded where others who had employed in vivoapproaches had failed. The principal reason for this is that the unidentified intermediates in the tryptophan pathway are all phosphorylated. Phosphorylated intermediates accumulated in vivo would have been dephosphorylated and therefore inactive when fed to a mutant. With the aid of my graduate student Oliver Smith, the following intermediates were identified: phosphoribosyl anthranilate, carboxyphenylamino-1-deoxyribulose 5-phosphate, and indole-3-glycerol phosphate (IGP). The initial precursor of the tryptophan pathway, chorismic acid, was isolated and identified by Frank Gibson, working with his own group in Australia. Chorismate also serves as precursor of the other aromatic amino acids. With the identification of these additional compounds, the precursor and all the intermediates in the tryptophan biosynthetic pathway were known. While conducting these studies I made an unanticipated observation that subsequently proved to be of enormous benefit in our colinearity studies. I observed that many tryptophan auxotrophs of E. coli, when cultured on growth-limiting levels of tryptophan, produced 20–50 times more tryptophan synthetase than the wild type strain. I thought that the day might come, as it did, when I could exploit this observation to overproduce mutant proteins for purification and analysis. I was aware of the regulatory significance of this observation and concluded that ultimately we should address the regulatory mechanism(s) responsible for this increase. Despite this temporary diversion in the mid-1950s, I was still committed to establishing the nature of the gene-enzyme relationship. Knowledge about genes, proteins, and protein synthesis was improving, so much so that the gene-enzyme relationship was redefined. The question had matured to the following. Is the nucleotide sequence of a gene colinear with the amino acid sequence of the corresponding protein? During this period we learned many new facts about tryptophan synthetase. I thought it might prove to be an ideal enzyme for addressing the colinearity question. Our continuing investigations with this enzyme, from both Neurospora and E. coli, suggested that it may catalyze the last two reactions in tryptophan formation, the cleavage of IGP to indole and the coupling of indole with serine to form tryptophan. However, there were two observations we could not explain: free indole could not be detected as an intermediate in the conversion of IGP to tryptophan, and the rate of conversion of IGP to indole was lower than its rate of conversion to tryptophan (14Yanofsky C. Rachmeler M. The exclusion of free indole as an intermediate in the biosynthesis of tryptophan in Neurospora crassa..Biochim. Biophys. Acta. 1958; 28: 641-642Google Scholar). We then had to ask the following question. Does the enzyme catalyze a third reaction in which IGP and serine react with one another to form tryptophan, or is indole truly the intermediate, and it remains within the enzyme complex? This puzzle was not satisfactorily solved until the late 1980s. Then, the elegant structural solution for the α2β2 tryptophan synthase (name changed again) enzyme complex of Salmonellaby Hyde et al. (15Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. Three-dimensional structure of the tryptophan synthase α2β2 multienzyme complex from Salmonella typhimurium..J. Biol. Chem. 1988; 263: 17857-17871Google Scholar) revealed that there is a physical tunnel in this enzyme complex connecting the active site of one polypeptide subunit, α, where indole is produced from IGP, to an active site of the second subunit, β2, where indole reacts withl-serine to form l-tryptophan (15Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. Three-dimensional structure of the tryptophan synthase α2β2 multienzyme complex from Salmonella typhimurium..J. Biol. Chem. 1988; 263: 17857-17871Google Scholar, 16Miles E.W. Biswas B.B. Roy S. Tryptophan synthase: structure, function, and protein engineering. In Subcellular Biochemistry, Proteins: Structure, Function, and Protein Engineering. 24. Plenum Press, New York1995: 207-254Google Scholar). As you might imagine, it was comforting to have our confusing early observations explained unambiguously by structural and enzymatic studies. At this stage in my career everything was going well for me at Western Reserve Medical School. I had quality co-workers and I thoroughly enjoyed my interactions with my fellow faculty members, Howard Gest, John Spizizen, David Novelli, Bob Greenberg, and Abe Stavitsky. However, in 1957 I was contacted by Victor Twitty, chairman of the Department of Biological Sciences at Stanford University, and offered a faculty position. Despite my initial disinterest in considering this appointment, I accepted their offer for a variety of reasons, including my learning that Arthur Kornberg's department would be moving to the Stanford campus (to the Stanford Medical School, which was being relocated from San Francisco). Of historical interest, when I arrived at Stanford the laboratory space I was provided was in the basement of old Jordan Hall and was the space previously occupied by Ed Tatum and his research team. I truly was treading in Tatum's footsteps! When setting up my laboratory at Stanford in January of 1958, I decided that the time had come to mount an all out effort to establish or disprove gene-protein colinearity. I was joined in this project by an outstanding young postdoctoral fellow, Irving Crawford, who was recommended to me by Arthur Kornberg. In his exploratory studies with tryptophan synthetase from E. coli, Irving was first to establish that the enzyme is a complex composed of non-identical polypeptide chains. One subunit, TrpA (TSase α), hydrolyzes IGP to indole, whereas the second subunit, TrpB (TSase β2), covalently joins indole and l-serine to forml-tryptophan (17Crawford I.P. Yanofsky C. On the separation of the tryptophan synthetase of Escherichia coli into two protein components..Proc. Natl. Acad. Sci. U. S. A. 1958; 44: 1161-1170Google Scholar). However, the enzyme fromNeurospora is a single polypeptide chain. In what proved to be an extremely valuable observation for our subsequent colinearity studies, Irving found that each E. coli subunit activates the other subunit in the reaction that subunit performs alone. This finding suggested that we might be able to detect and assay each inactive TrpA mutant protein enzymatically by measuring its ability to activate the TrpB subunit in the indole plus serine to tryptophan reaction. This expectation proved to be correct; we routinely assayed each mutant TrpA protein during its purification by measuring its activation of TrpB. We next prepared a set of pure mutant TrpA proteins, each presumably with a single inactivating amino acid change. Good fortune helped us again, for in 1958 Vernon Ingram described an elegant method, "peptide fingerprinting," which he had used to detect peptides with single amino acid changes in mutant human hemoglobins (18Ingram V.M. Abnormal human hemoglobins. 1. The comparison of normal human and sickle-cell hemoglobins by fingerprinting..Biochim. Biophys. Acta. 1958; 28: 539-545Google Scholar). This approach seemed ideal for what we wished to do. If we could identify the single amino acid change in each of our mutant proteins we would then only have to compare the positions of these amino acid changes in TrpA with the order of the corresponding altered sites on a fine structure genetic map of the trpA gene to prove or disprove gene-protein colinearity. I knew that we could construct a fine structure genetic map of trpA using phage P1, based on a previous genetic study I performed with Ed Lennox (19Yanofsky C. Lennox E.S. Transduction and recombination study of linkage relationships among the genes controlling tryptophan synthesis in Escherichia coli..Virology. 1958; 8: 425-447Google Schol