GDP-mannose mannosyl hydrolase catalyzes nucleophilic substitution at carbon, unlike all other nudix hydrolases

Abstract

GDP-mannose mannosyl hydrolase (GDPMH) from Escherichia coli is a 36.8 kDa homodimer which, in the presence of Mg2+, catalyzes the hydrolysis of GDP-α-D-mannose or GDP-α-D-glucose to yield sugar and GDP. On the basis of its amino acid sequence, GDPMH is a member of the Nudix family of enzymes which catalyze the hydrolysis of nucleoside diphosphate derivatives by nucleophilic substitution at phosphorus. However, GDPMH has a sequence rearrangement (RE to ER) in the conserved Nudix motif and is missing a Glu residue characteristic of the Nudix signature sequence. By 1H NMR, the initial hydrolysis product of GDP-α-D-glucose is β-D-glucose, indicating nucleophilic substitution with inversion at C1' of glucose. Substitution at carbon was confirmed by two-dimensional 1H-13C HSQC spectra of the products of hydrolysis in 48.4% 18O-labeled water which showed an additional C1' resonance of β-D-glucose with a typical upfield 18O isotope shift of 18 ppb and an intensity of 47.6% of the total signal. No 18O isotope-shifted resonances (<4%) were found in the 31P NMR spectrum of the GDP product. Thus, unlike all other Nudix enzymes studied so far, GDPMH catalyzes nucleophilic substitution at carbon rather than at phosphorus. A small solvent kinetic deuterium isotope effect on k(cat) of 1.76 ±0.25, independent of pH over the range of 6.0-9.3, suggests that the deprotonation of water may be part of the rate-limiting step.