Discoveries in bacterial nucleotide metabolism.

My decision to become a biochemist has its roots in youthful indecision. I grew up on a dairy farm in northwestern Illinois surrounded by domestic and wild animal and plant life and came to love the world of biology. This interest was strongly encouraged by my mother, who had been an excellent student of biology in college but was thwarted by the Great Depression and her family's poverty from pursuing an opportunity for graduate study. Her books, preserved specimens, and microscope slides became my playthings. I set up a home “lab” using commercial chemistry sets from Gilbert and ChemCraft, items that are regrettably deemed unsafe for sale today, but I did not gain a good understanding of chemical principles until high school. There I became enamored of the ability of chemistry to explain the properties of matter, including living matter, in rigorous and elegant atomic and molecular terms. How to compromise between these two fascinating fields of science? I decided in high school to seek a mixture, biochemistry, even though I had no real understanding of what that discipline involved. It proved to be an excellent decision! As the state's land grant university, the University of Illinois at Urbana was a natural (and the only affordable) choice for my undergraduate education, especially after I won a scholarship by competitive examination that paid tuition and fees for all four years (1957–1961). Only gradually did I discover my good fortune: the University of Illinois had one of the most distinguished chemistry departments in the United States, and I received an excellent, intensive education. Biochemistry was deemed a graduate specialty at the time, so I majored in chemistry but took biochemistry and microbiology lecture and lab courses as soon as I could. Senior research under the direction of Carl Vestling, who left me largely on my own, introduced me to the confusions and ultimate satisfactions of research. I had the good fortune to win a National Science Foundation fellowship for graduate study, and I enrolled at the University of California at Berkeley, which was recognized as having one of the nation's finest departments of biochemistry. I had no idea of the specialty I wished to pursue, so I was attracted to the breadth and depth of research topics pursued by Berkeley's outstanding faculty. My graduate school years from 1961 to 1966 were wonderfully exciting years for a young biochemist! The genetic code was being cracked. The groundbreaking papers by Jacob and Monod on regulatory genes were the subject of intense discussion: could such powerful biochemical conclusions be based on genetics alone? The concept of the subunit structure of proteins was being clarified, and the first high-resolution x-ray structures of proteins were emerging. I chose to pursue Ph.D. thesis research under H. A. Barker because I was fascinated with his recent discovery of the coenzyme form of vitamin B12 and the fact that no one understood the chemical basis for its role in catalysis. Barker was a modest, gentle man who emphasized the importance of rigorous, critical experimentation. Initially, he observed my work carefully, but gradually he granted me freedom to design and interpret experiments quite independently. We were able to make substantial contributions to the enzymology of the coenzyme B12-dependent glutamate mutase system (1), but the most important contemporary contribution to the mechanism of action of the coenzyme came from the work of Perry Frey and Robert Abeles, who demonstrated the transfer of hydrogen atoms between substrates and the 5′-methylene of the [Co]-deoxyadenosyl moiety of the coenzyme (2). We later showed that the same remarkable chemistry occurred in the glutamate mutase reaction (3). From Barker, I learned an important lesson: investigation of an experimental system of seemingly narrow or specialized interest, such as anaerobic fermentations in the clostridia, could lead, if pursued carefully, thoughtfully, and with an alert eye for the unexpected, to discoveries of very great general importance. Herb Weissbach made this same point in his recent Reflections article (4). With respect to my independent research career, one might have thought that the biochemistry of nucleotide biosynthesis was well understood when I began my studies of it, but we were richly rewarded with novel findings. With Barker's encouragement, I applied in 1966 to Earl Stadtman for postdoctoral studies at the National Institutes of Health (NIH). Again, I had the good fortune to win a fellowship, this time from the NIH, for support of my research training. By doing research with Earl Stadtman (and with Terry Stadtman, who worked in the same laboratory and was an excellent source of intellectual and methodological advice), I was staying in Barker's scientific family; both Stadtmans had trained with Barker and emphasized his style of critical, evidence-driven research, typically with bacterial systems. My work with Barker emphasized metabolic pathways and enzymology, which were central to the biochemistry of the time, but Stadtman opened my eyes to the regulation of metabolism. An array of clever mechanisms selected through evolution for regulation of enzymes was already known, and new discoveries frequently appeared. My fascination with the regulation of enzyme activity and gene expression has continued to yield significant discoveries for 40 years. It would be difficult to imagine a laboratory more exciting than the Stadtman lab during the years I worked there (1966–1968) (Fig. 1). Kingdon and Shapiro discovered the regulation of glutamine synthetase by adenylylation; Ron Kaback was studying the mechanism of active transport; and others were investigating the mechanism of coenzyme B12 action, the biochemistry of methane formation, and other pathways of bacterial metabolism. An atmosphere of excitement and intense scientific debate prevailed. The “Stadtman way” of mentoring the development of scientists has been deservedly recognized (5). Good research ideas came readily to Earl, and he shared them unselfishly with his postdoctoral students, encouraging them to develop independent research themes from them; often, as he did with me, he declined even to add his name to the resulting publications. By today's standards, a postdoctoral research period of only two years is too short to qualify one for an independent faculty position, but I had the good fortune in 1968 to be offered an assistant professorship in biochemistry at my alma mater, Illinois. I jumped at the opportunity and have spent my entire career there. FIGURE 1. The Stadtman laboratory (officially called the Laboratory of Biochemistry of the National Heart Institute) in 1966. Terry and Earl Stadtman are second and third from the left, respectively, in the second row; I am second from the left in the back row. ...