Visualization of mutagenic nucleotide processing by Escherichia coli MutT, a Nudix hydrolase

Escherichia coli MutT prevents mutations by hydrolyzing mutagenic 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) in the presence of Mg(2+) or Mn(2+) ions. MutT is one of the most studied enzymes in the nucleoside diphosphate-linked moiety X (Nudix) hydrolase superfamily, which is widely distributed in living organisms. However, the catalytic mechanisms of most Nudix hydrolases, including two- or three-metal-ion mechanisms, are still unclear because these mechanisms are proposed using the structures mimicking the reaction states, such as substrate analog complexes. Here, we visualized the hydrolytic reaction process of MutT by time-resolved X-ray crystallography using a biological substrate, 8-oxo-dGTP, and an active metal ion, Mn(2+). The reaction was initiated by soaking MutT crystals in a MnCl(2) solution and stopped by freezing the crystals at various time points. In total, five types of intermediate structures were refined by investigating the time course of the electron densities in the active site as well as the anomalous signal intensities of Mn(2+) ions. The structures and electron densities show that three Mn(2+) ions bind to the Nudix motif of MutT and align the substrate 8-oxo-dGTP for catalysis. Accompanied by the coordination of the three Mn(2+) ions, a water molecule, bound to a catalytic base, forms a binuclear Mn(2+) center for nucleophilic substitution at the β-phosphorus of 8-oxo-dGTP. The reaction condition using Mg(2+) also captured a structure in complex with three Mg(2+) ions. This study provides the structural details essential for understanding the three-metal-ion mechanism of Nudix hydrolases and proposes that some of the Nudix hydrolases share this mechanism.