A Trail of Research on Cofactors: An Odyssey with Friends

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 Zn2+ with the eucaryotic enzymes and delimited effects of inhibitors that relate to drug action. Flavin adenine dinucleotide synthetase, separable from flavokinase in mammals, prefers Mg2+. Specifics for binding and function of flavocoenzymes were delineated for several flavoproteins. The flavin mononucleotide-dependent oxidase that converts the 5'-phosphates of pyridoxine and of pyridoxamine to pyridoxal 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.