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

Abstract

Significance Time-resolved X-ray crystallography has captured transient elements in enzymatic reactions and has advanced understanding regarding canonical mechanisms. Although Nudix hydrolases are involved in cellular metabolism via hydrolytic activities, the catalytic mechanisms remain elusive. This is because the Nudix hydrolases are defined only by a highly conserved short motif and because most mechanisms are based on the structures mimicking the reaction states. Time-resolved X-ray crystallography of MutT has followed the course of 8-oxo-dGTP hydrolysis and enabled visualization of the process of nucleophilic substitution by a water molecule that is synchronized with the sequential coordination of three metal ions. These observations are consistent with previous kinetic analysis and are indicative of a three-metal-ion mechanism.

Protonation states of Asp residues in the human Nudix hydrolase MTH1 contribute to its broad substrate recognition

“…These results indicate that the loss of activity of the wild‐type at higher pH is mainly caused by the inability of substrate binding (Fig. 3), whereas the reduced activity of the D120N mutant at higher pH is caused by the impairment of the base catalyst Glu52, whose function is suggested by experiments with E. coli counterpart MutT [20,36] (Fig. S7) because the D120N mutant recognizes 2‐oxo‐dATP even at higher pH (Fig.…”

“…Five types of the crystal structures of the MTH1-2-oxo-dATP complex at pH 7.7-9.7 were determined at 1.05-1. 20 A resolutions (Fig. 3, Table S1).…”

“…One of the characteristic features of the enzymatic activity of MTH1 is its broad substrate specificity for oxidized dNTPs, such as 8‐oxo‐dGTP and 2‐oxo‐dATP [4,5], which is completely different from the extremely high substrate specificity of Escherichia coli counterpart MutT for 8‐oxo‐dGTP [16–20]. The solution structure of the MTH1 apo form determined using NMR and kinetic analyses revealed that Asp119, Asn33, Phe27, and Trp117 at the substrate‐binding site contribute differently to the recognition of 8‐oxo‐dGTP and 2‐oxo‐dATP [21,22].…”

Visualizing the three-metal-ion-dependent cleavage of a mutagenic nucleotide

“…Kinetic and static structural studies of MutT show that the hydrolytic reaction may proceed via a two-metal-ion mechanism ( 1 , 3 – 7 ). However, differences in substrate binding, metal coordination, and number of metals observed in the active site among the published structures leave unanswered questions about the MutT reaction pathway that are addressed by Nakamura and Yamagata in PNAS ( 8 ).…”

“…Nakamura and Yamagata resolve these conflicts by using in crystallo catalysis and time-lapse crystallography to observe MutT catalyzed hydrolysis of the wild-type enzyme and its genuine substrate, the mutagenic nucleotide 8-oxo-dGTP, in the presence of divalent metal cations Mn 2+ or Mg 2+ ( 8 ). The enzyme–substrate complex was initially cocrystallized in the absence of Mg 2+ /Mn 2+ and the reaction was initiated by soaking the crystals in solutions containing divalent cations at concentrations that support catalysis for discrete time periods and stopped by freezing ( 12 ).…”