Symposium: Mechanistic Aspects of Vitamin and Coenzyme Utilization and Function: A Symposium in Recognition of the Distinguished Career of Donald B. McCormick A Trail of Research on Cofactors: An Odyssey with Friends1 Donald B. McCormick Department of Biochemistry, Rollins Research Center, Emory University, Atlanta, GA 30322-3050 Downloaded from https://academic.oup.com/jn/article/130/2/323S/4686386 by guest on 30 September 2021 ABSTRACT Over the span of 40 y and with the participation of over 60 students and postdoctoral colleagues, my laboratory has been able to elucidate numerous aspects of cofactor metabolism and function. Findings have been on the absorption, transport, utilization and excretion of vitamin B-6, riboflavin, biotin, lipoate and ascorbate. Specificity studies on those trace but essential enzymes that catalyze conversion of such vitamins as B-6 and riboflavin to their functional coenzymes led to our development of “biochemically specific absorbents” that prototypically exemplified what later was called “affinity chromatography.” Characterization of the purified kinases for B-6 and riboflavin revealed preference for Zn2ϩ with the eucaryotic enzymes and delimited effects of inhibitors that relate to drug action. Flavin adenine dinucleotide synthetase, separable from flavokinase in mammals, prefers Mg2ϩ. Specifics for binding and function of flavocoenzymes were delineated for several flavoproteins. The flavin mononucleotide-dependent oxidase that converts the 5Ј-phosphates of pyridoxine and of pyridoxamine to pyri- doxal phosphate is a connection between riboflavin and B-6 that we characterized in mechanistic detail and found to be the primary control point for conversion of B-6 to its coenzyme. Sequencing and cloning of a side-chain oxidase for riboflavin was achieved. Isolation and identification of metabolites of biotin and of lipoic acid, first from bacteria obtained by enrichment culture and then from mammals, provided seminal information on catabolic pathways involved, as have our other studies with flavin catabolites isolated from milk and urine. J. Nutr. 130: 323S–330S, 2000. KEY WORDS: ● cofactors ● water-soluble vitamins ● coenzymes ● divalent cations ● vitamin metabolism Although it is relatively easy to summarize one’s own re- extension of glucuronate metabolism is toward ascorbate (vi- search accomplishments, it is difficult to present them without tamin C) as well as xylulose, and there was a strong interest in the use of first person and seeming to be self-serving. Yet this nutritional biochemistry within the Vanderbilt department whole exercise should be to emphasize the partnerships that when Bill Darby was chairman, my polarized interests were to make science. Hence, I have attempted to list within each of head west where there were strong elements of vitamin/coen- the following subdivisions of my research topics the names of zyme research at the University of California-Berkeley. colleagues who worked with me, and to use sufficient citations to literature to document our experimental findings. My doctoral dissertation research, mentored by Professor Vitamin B-6 metabolism Oscar Touster at Vanderbilt, was on pentose and pentitol metabolism. Studies on isotopically labeled pentitols and pen- Pyridoxal (pyridoxine, pyridoxamine) kinase—M. Greg- toses helped establish the metabolic interrelationships of xy- ory, E. Snell. During postdoctoral research with Professor Es- litol/xylulose to glucose/glucuronate (Touster et al. 1957) and mond Snell at Berkeley, I began work initiated by M. Gregory. further to the pentose phosphate pathway (McCormick and Isolation and comparative studies on both pro- and eucaryotic forms of pyridoxal kinase delineated general properties, includ- Touster 1957). The general interdigitations of pentitol metab- 2ϩ 2ϩ olism were extended (McCormick and Touster 1961) and the ing the first substantiated role of Zn in preference to Mg genetic defect of essential pentosuria clarified. Because one as the cosubstrate ATP complex for the mammalian phos- phokinase (McCormick et al. 1961), and led to circumscrip- tion of inhibitory aspects (McCormick and Snell 1961), in- 1 Presented as part of the symposium “Mechanistic Aspects of Vitamin and cluding the potent action of carbonyl reagents (McCormick Coenzyme Utilization and Function: A Symposium in Recognition of the Distin- 1959, McCormick et al. 1960, McCormick and Snell 1959) guished Career of Donald B. McCormick” as part of the Experimental Biology 99 and such drugs as are known to bind to the kinase (McCor- meeting held April 17–21 in Washington, D.C. This symposium was sponsored by the American Society for Nutritional Sciences. The proceedings of this sympo- mick and Chen 1999). .sium are published as a supplement to Guest editors for this supplement publi- Pyridoxine (pyridoxamine) 5؅-phosphate oxidase—P cation were Alfred H. Merrill, Jr., Emory University School of Medicine, Atlanta, Barsa, D. Bowers-Komro, H. Chen, J. Choi, M. Davis, M. GA; Barbara B. Bowman, U.S. Centers for Disease Control and Prevention, Atlanta, GA; and Peter C. Preusch, National Institutes of General Medical Sci- DePecol, D. Edmondson, K. Horiike, S. Kasai, M. Kazarinoff, ences, Bethesda, MD. W. Korytnyk, K. Matsui, A. Merrill, K. Ohashi, K. Rasmussen, 0022-3166/00 $3.00 © 2000 American Society for Nutritional Sciences. 323S 324S SUPPLEMENT D. Roe, H. Tsuge, K. Watanabe. As a faculty member at Cornell and Emory with the help of graduate and postdoctoral coworkers, we succeeded in the first complete purification of pyridoxine (pyridoxamine) 5Ј-phosphate oxidase, the flavin mononucleotide (FMN)2-dependent enzyme responsible for conversion of the kinase-derived phosphovitamin B-6 to co- enzymic pyridoxal 5Ј-phosphate (Kazarinoff and McCormick 1975). More facile affinity purifications (Bowers-Komro et al. 1986, Tsuge and McCormick 1980) and assays (DePecol and McCormick 1980) were developed and circumscription of substrate (DePecol and McCormick 1980, Bowers-Komro and McCormick 1987, Kazarinoff and McCormick 1973, Ka- zarinoff and McCormick 1975, Merrill et al. 1980) and coen- zyme specificities (Kazarinoff and McCormick 1974, Merrill et Downloaded from https://academic.oup.com/jn/article/130/2/323S/4686386 by guest on 30 September 2021 al. 1979b) accomplished. Systematic elucidation of the dimeric subunit association (Horiike et al. 1979a, Tsuge and McCormick 1980), active-site amino acid residues (Bowers- Komro et al. 1986, Choi and McCormick 1981, Horiike et al. 1979b, McCormick et al. 1976, Tsuge and McCormick 1980), kinetics (Choi et al. 1982, Choi et al. 1983), and ultimately FIGURE 1 Interconversions of pyridoxine (PN), pyridoxal (PL) and mechanistic delineation of stereochemical aspects (Bowers- pyridoxamine (PM) with their 5Ј-phosphates (PNP, PLP, PMP) as cat- Komro and McCormick 1984b, Bowers-Komro and McCor- alyzed by pyridoxal kinase (K), phosphatase (P) and pyridoxine (pyri- mick 1985a, McCormick and Bowers-Komro 1986) have pro- doxamine) 5Ј-phosphate oxidase (O). vided definitive information on the way this essential flavoprotein operates (Bowers-Komro and McCormick 1984a), depends upon flavin status of an organism (Rasmussen et al. Cormick 1964b, McCormick et al. 1997) and accomplished its 1979, Rasmussen et al. 1980), and participates in the regula- partial (Gomes and McCormick 1983) and then complete tion of B-6 metabolism (McCormick and Merrill 1980, Merrill purification, again using affinity (FMN-agarose) methods (Oka et al. 1978b). The sequences for this essential oxidase from and McCormick 1987). Further work led to more detailed several organisms have been determined (McCormick and characterization of the cooperatively interactive kinase/syn- Chen 1999). An important interface between vitamins B-2 thetase system and to their kinetic mechanisms (Yamada et al. and B-6 is now clear. 1990). The scheme given in Figure 1 outlines the sequential roles FMN phosphatase and FAD pyrophosphatase—S. Lee, of kinase and oxidase in the interconversions of B-6 vitamers M. Russell. The interfering, nonspecific actions of alkaline toward the coenzyme pyridoxal 5Ј phosphate. and acid FMN phosphatases (McCormick 1961, McCormick and Russell 1962) and FAD pyrophosphatase have been sep- Flavin metabolism arated and generally characterized as degradative hydrolases responsible for breakdown of flavocoenzymes (Lee and McCor- Flavokinase—C. Arsenis, R. Butler, B. Chassey, P. Hem- mick 1983). merich, S. Lee, A. Merrill, H. Nakano, Y. Yamada, Z. Zak. The Riboflavin side-chain oxidases—H. Chen, D. Edmondson, first significant purification of flavokinase, shown by us to be T. Kekelidze, C. Yang. A bacterial side-chain oxidizing en- another Zn2ϩ preferring enzyme (McCormick 1962, Merrill zyme that had been called a “hydrolase” was found by us to and McCormick 1980, Nakano and McCormick 1991b) re- have relative specificity (Yang and McCormick 1967a), sponsible for catalyzing phosphorylation of riboflavin to yield whereas another enzyme narrowly specific for riboflavin FMN, was accomplished with classic techniques (McCormick (Kekelidze et al. 1994, Kekelidze et al. 1995) has been molec- 1962) and completely purified from mammalian tissues by ularly cloned and sequenced by us from a fungal organism “affinity techniques” (Arsenis and McCormick 1964a, Merrill (Chen and McCormick 1997a) and found able to form both and McCormick 1980, Nakano and McCormick 1991a) before aldehyde and acid products at the 5Ј-terminus (Chen and the term and practice became commonplace. Detailed studies McCormick 1997b). on the specificity