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day. The ADPR complex crystals were then transferred into Zn ion solution for starting hydrolysis. After eight kinds of reaction times from 3 min to 1 hr, each crystal was cryo-trapped in N2 gas stream at 100 K and diffraction data was collected independently over 1.6 Å resolution. The crystals were all highly isomorphous to the initial ADPR complex one. Nine structures were refined by REFMAC in the CCP4 program suits to the final R-values less than 20 %. Results clearly show that (i) ADPR in the reaction cavity changes its conformation to a reaction intermediate within 10-15 min, (ii) a water molecule coordinated to the Zn ion at the M-I site is activated to hydroxide (OH) by Glu82, and (iii) the OH anion attacks the α -phosphorus atom of ADPR in the inline geometry to the removing oxygen between α-P and β-P. The real pathway revealed is different from both of the mechanisms previously proposed. It is indicated once again that reaction mechanisms based on 3D crystallography should be proofed by the time-resolved analysis.

Ligand-induced conformational change of D-alanine:D-alanine ligase fromThermus thermophilusHB8