Four-dimensional structure analysis of Ndx family enzyme utilizing a new pH-temperature jump trigger

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Before starting our research, we tried to improve crystal quality of ADP-ribose pyro-phosphatase (ADPRase) involved in Ndx family, because we needed high resolution analysis to resolve substrates and products which would co-exist in the reaction cavity of ADPRase in time course of enzyme reaction. Many crystallization conditions were tested, and the observable diffraction limit was expanded to 1.1A resolution from 1.7A, previously reported. In order to start the crystalline state reaction of ADPRase, Zn(II) ions were soaked into the crystals, in which the substrate ADPR was introduced in advance. The reaction was stopped by a temperature jump from 295K to 90K. Crystal structures were determined at 9 points of reaction time, 0, 3, 6, 10, 15, 20, 30, 40, 60min, and the in situ observation of ADPRase reaction was succeeded. Our results showed that the enzyme reaction progressed in crystal after the Zn(II) ion soaking as following. (1) The first Zn(II) ion introduced into reaction cavity changed ADPR conformation to an intermediate state, designated as ADPR^*.(2) The second Zn(II) ion ligated ADPR^*, gultamate side chain of E82 and E86, and two water molecules making a five-ligands coordination structure. (3) One of two water molecules was activated by the second Zn(II) ion and E82 to hydroxide anion, and (4) the anion attacked nucleophilically the alpha phosphate of ADPR to brake the pyrophosphate bond to produce two products, AMP and ribose-5'-phosphate. The biochemical insight into the in situ observation of ADPRase was discussed.