Abstract
Spore photoproduct lyase (SPL) catalyzes the direct reversal of a thymine dimer 5-thyminyl-5,6-dihydrothymine (i.e. the spore photoproduct (SP)) to two thymine residues in germinating endospores. Previous studies suggest that SPL from the bacterium Bacillus subtilis (Bs) harbors an unprecedented radical-transfer pathway starting with cysteine 141 proceeding through tyrosine 99. However, in SPL from the bacterium Clostridium acetobutylicum (Ca), the cysteine (at position 74) and the tyrosine are located on the opposite sides of a substrate-binding pocket that has to collapse to bring the two residues into proximity, enabling the C→Y radical passage as implied in SPL(Bs) . To test this hypothesis, we adopted hydrogen/deuterium exchange mass spectrometry (HDX-MS) to show that C74(Ca) is located at a highly flexible region. The repair of dinucleotide SP TpT by SPL(Ca) is eight-fold to 10-fold slower than that by SPL(Bs) ; the process also generates a large portion of the aborted product TpTSO2- . SPL(Ca) exhibits apparent (D V) kinetic isotope effects (KIEs) of ~6 and abnormally large competitive (D V/K) KIEs (~20), both of which are much larger than the KIEs observed for SPL(Bs) . All these observations indicate that SPL(Ca) possesses a flexible active site and readily undergoes conformational changes during catalysis.