Enzyme Activation with a Synthetic Catalytic Co‐enzyme Intermediate: Nucleotide Methylation by Flavoenzymes. Issue 41 (5th September 2017) - Bou‐Nader, Charles
Abstract: 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. Abstract : Artificial enzyme engineering is used to uncover cofactor‐dependent complex mechanistic issues. A chemically synthesized N5‐flavin iminium catalytic intermediate can be used to artificially activate the apoprotein version of ThyX and TrmFO for dUMP and tRNA C5 uracil methylation. This procedure bypasses the natural biosynthetic pathway and establishes this flavin iminium species as the genuine nucleotide methylating agent.