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

Diadenosine polyphosphates (Apn A, n=2-6), particularly Ap4 A, 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 Ap4 A hydrolase suggested that MsMutT1 could also hydrolyse diadenosine polyphosphates. Biochemical experiments yielded results in conformity with this suggestion, with Ap4 A as the best among the substrates. ATP is a product in all experiments; small amounts of ADP were also observed in the experiments involving Ap4 A and Ap6 A. Hydrolysis was inhibited by fluoride ions in all cases. The mechanism of action and its inhibition in relation to Apn A were explored through the X-ray analysis of the crystals of the MsMutT1 complexes with Ap5 A; Ap5 A and MnCl2 ; Ap4 A; ATP; and ATP.NaF.MgCl2 . 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 Apn A 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.