Hydrolysis of diadenosine polyphosphates. Exploration of an additional role of Mycobacterium smegmatis MutT1.

Diadenosine polyphosphates (ApA, n=2-6), particularly ApA, are involved in several important physiological processes. The substantial sequence identity of the Nudix hydrolase domain (domain 1) of Mycobacterium smegmatis MutT1 (MsMutT1) with a known ApA hydrolase suggested that MsMutT1 could also hydrolyse diadenosine polyphosphates. Biochemical experiments yielded results in conformity with this suggestion, with ApA as the best among the substrates. ATP is a product in all experiments; small amounts of ADP were also observed in the experiments involving ApA and ApA. Hydrolysis was inhibited by fluoride ions in all cases. The mechanism of action and its inhibition in relation to ApA were explored through the X-ray analysis of the crystals of the MsMutT1 complexes with ApA; ApA and MnCl; ApA; ATP; and ATP.NaF.MgCl. The aggregation pattern of molecules in the first four crystals is similar to that found in a majority of MsMutT1-NTP crystals. Substrate molecules occupy the primary binding site and ATP occupies a site at an intermolecular interface, in the first two. ATP occupies both the sites in the third and fourth crystal. The protein-ligand interactions observed in these crystal structures lead to an explanation of the molecular mechanism of hydrolysis of ApA by MsMutT1. The fifth crystal exhibits a new packing arrangement. The structure of the complex provides an explanation for the fluoride inhibition of the activity of the enzyme. It would thus appear that MutT1 has a major role involving the hydrolysis of diadenosine polyphosphates, which could be elucidated at the molecular level.