To facilitate production of functional enzymes and to study their mechanisms, especially in the complex cases of coenzyme-dependent systems, activation of an inactive apoenzyme preparation with a catalytically competent coenzyme intermediate is an attractive strategy. This is illustrated with the simple chemical synthesis of a flavin-methylene iminium compound previously proposed as a key intermediate in the catalytic cycle of several important flavoenzymes involved in nucleic acid metabolism. Reconstitution of both flavin-dependent RNA methyltransferase and thymidylate synthase apoproteins with this synthetic compound led to active enzymes for the C5-uracil methylation within their respective transfer RNA and dUMP substrate. This strategy is expected to be of general application in enzymology.