Deoxyhypusine synthase generates and uses bound NADH in a transient hydride transfer mechanism

Deoxyhypusine is a modified lysine residue. It is formed posttranslationall y in the precursor of eukaryotic initiation factor 5A (eIF5A) by deoxyhypus ine synthase, employing spermidine as a butylamine donor. In the initial st ep of this reaction, deoxyhypusine synthase catalyzes the production of NAD H through dehydrogenation of spermidine. Fluorescence measurements of this reaction revealed a -22-nm blue shift in the emission peak of NADH and a si milar to 15-fold increase in peak intensity, characteristics of tightly bou nd NADH that were not seen by simply mixing NADH and enzyme. The fluorescen t properties of the bound NADH can be ascribed to a hydrophobic environment and a rigidly held, open conformation of NADH, features in accord with the known crystal structure of the enzyme. Considerable fluorescence resonance energy transfer from tryptophan 327 in the active site to the dihydronicot inamide ring of NADH was seen. Upon addition of the eIF5A precursor, utiliz ation of the enzyme-bound NADH for reduction of the eIF5A-imine intermediat e to deoxyhypusine was reflected by a rapid decrease in the NADH fluorescen ce, indicating a transient hydride transfer mechanism as an integral part o f the reaction. The number of NADH molecules bound approached four/enzyme t etramer; not all of the bound NADH was available for reduction of the eIF5A -imine intermediate.