Publications

• X-ray, NMR, and mutational studies of the catalytic cycle of the GDP-mannose mannosyl hydrolase reaction.

  Gabelli S.B., Azurmendi H.F., Bianchet M.A., Amzel L.M., Mildvan A.S.

  GDP-mannose hydrolase catalyzes the hydrolysis with inversion of GDP-alpha-D-hexose to GDP and beta-D-hexose by nucleophilic substitution by water at C1 of the sugar. Two new crystal structures (free enzyme and enzyme-substrate complex), NMR, and site-directed mutagenesis data, combined with the s ... >> More

  GDP-mannose hydrolase catalyzes the hydrolysis with inversion of GDP-alpha-D-hexose to GDP and beta-D-hexose by nucleophilic substitution by water at C1 of the sugar. Two new crystal structures (free enzyme and enzyme-substrate complex), NMR, and site-directed mutagenesis data, combined with the structure of the enzyme-product complex reported earlier, suggest a four-stage catalytic cycle. An important loop (L6, residues 119-125) contains a ligand to the essential Mg2+ (Gln-123), the catalytic base (His-124), and three anionic residues. This loop is not ordered in the X-ray structure of the free enzyme due to dynamic disorder, as indicated by the two-dimensional 1H-15N HMQC spectrum, which shows selective exchange broadening of the imidazole nitrogen resonances of His-124 (k(ex) = 6.6 x 10(4) s(-1)). The structure of the enzyme-Mg2+-GDP-mannose substrate complex of the less active Y103F mutant shows loop L6 in an open conformation, while the structure of the enzyme-Mg2+-GDP product complex showed loop L6 in a closed, "active" conformation. 1H-15N HMQC spectra show the imidazole N epsilon of His-124 to be unprotonated, appropriate for general base catalysis. Substituting Mg2+ with the more electrophilic metal ions Mn2+ or Co2+ decreases the pKa in the pH versus kcat rate profiles, showing that deprotonation of a metal-bound water is partially rate-limiting. The H124Q mutation, which decreases kcat 10(3.4)-fold and largely abolishes its pH dependence, is rescued by the Y103F mutation, which increases kcat 23-fold and restores its pH dependence. The structural basis of the rescue is the fact that the Y103F mutation shifts the conformational equilibrium to the open form moving loop L6 out of the active site, thus permitting direct access of the specific base hydroxide from the solvent. In the proposed dissociative transition state, which occurs in the closed, active conformation of the enzyme, the partial negative charge of the GDP leaving group is compensated by the Mg2+, and by the closing of loop L2 that brings Arg-37 closer to the beta-phosphate. The development of a positive charge at mannosyl C1, as the oxocarbenium-like transition state is approached, is compensated by closing the anionic loop, L6, onto the active site, further stabilizing the transition state. << Less

  Biochemistry 45:11290-11303(2006) [PubMed] [EuropePMC]

• GDP-mannose mannosyl hydrolase catalyzes nucleophilic substitution at carbon, unlike all other Nudix hydrolases.

  Legler P.M., Massiah M.A., Bessman M.J., Mildvan A.S.

  GDP-mannose mannosyl hydrolase (GDPMH) from Escherichia coli is a 36. 8 kDa homodimer which, in the presence of Mg(2+), catalyzes the hydrolysis of GDP-alpha-D-mannose or GDP-alpha-D-glucose to yield sugar and GDP. On the basis of its amino acid sequence, GDPMH is a member of the Nudix family of e ... >> More

  GDP-mannose mannosyl hydrolase (GDPMH) from Escherichia coli is a 36. 8 kDa homodimer which, in the presence of Mg(2+), catalyzes the hydrolysis of GDP-alpha-D-mannose or GDP-alpha-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 (1)H NMR, the initial hydrolysis product of GDP-alpha-D-glucose is beta-D-glucose, indicating nucleophilic substitution with inversion at C1' of glucose. Substitution at carbon was confirmed by two-dimensional (1)H-(13)C HSQC spectra of the products of hydrolysis in 48.4% (18)O-labeled water which showed an additional C1' resonance of beta-D-glucose with a typical upfield (18)O isotope shift of 18 ppb and an intensity of 47.6% of the total signal. No (18)O isotope-shifted resonances (<4%) were found in the (31)P 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. << Less

  Biochemistry 39:8603-8608(2000) [PubMed] [EuropePMC]

• Kinetic and magnetic resonance studies of the role of metal ions in the mechanism of Escherichia coli GDP-mannose mannosyl hydrolase, an unusual nudix enzyme.

  Legler P.M., Lee H.C., Peisach J., Mildvan A.S.

  Biochemistry 41:4655-4668(2002) [PubMed] [EuropePMC]

• Mutational, kinetic, and NMR studies of the mechanism of E. coli GDP-mannose mannosyl hydrolase, an unusual Nudix enzyme.

  Legler P.M., Massiah M.A., Mildvan A.S.

  GDP-mannose mannosyl hydrolase (GDPMH) is an unusual Nudix family member, which catalyzes the hydrolysis of GDP-alpha-D-mannose to GDP and the beta-sugar by nucleophilic substitution at carbon rather than at phosphorus (Legler, P. M., Massiah, M. A., Bessman, M. J., and Mildvan, A. S. (2000) Bioch ... >> More

  GDP-mannose mannosyl hydrolase (GDPMH) is an unusual Nudix family member, which catalyzes the hydrolysis of GDP-alpha-D-mannose to GDP and the beta-sugar by nucleophilic substitution at carbon rather than at phosphorus (Legler, P. M., Massiah, M. A., Bessman, M. J., and Mildvan, A. S. (2000) Biochemistry 39, 8603-8608). Using the structure and mechanism of MutT, the prototypical Nudix enzyme as a guide, we detected six catalytic residues of GDPMH, three of which were unique to GDPMH, by the kinetic and structural effects of site-specific mutations. Glu-70 (corresponding to Glu-57 in MutT) provides a ligand to the essential divalent cation on the basis of the effects of the E70Q mutation which decreased kcat 10(2.2)-fold, increased the dissociation constant of Mn2+ from the ternary E-Mn2+-GDP complex 3-fold, increased the K(m)Mg2+ 20-fold, and decreased the paramagnetic effect of Mn2+ on 1/T1 of water protons, indicating a change in the coordination sphere of Mn2+. In the E70Q mutant, Gln-70 was shown to be very near the active site metal ion by large paramagnetic effects of Mn2+ on its side chain -NH2 group. With wild-type GDPMH, the effect of pH on log(kcat/K(m)GDPmann) at 37 degrees C showed an ascending limb of unit slope, followed by a plateau yielding a pK(a) of 6.4, which increased to 6.7 +/-0.1 in the pH dependence of log(kcat). The general base catalyst was identified as a neutral His residue by the DeltaH(ionization) = 7.0 +/-0.7 kcal/mol, by the increase in pK(a) with ionic strength, and by mutation of each of the four histidine residues of GDPMH to Gln. Only the H124Q mutant showed the loss of the ascending limb in the pH versus log(kcat) rate profile, which was replaced by a weak dependence of rate on hydroxide concentration, as well as an overall 10(3.4)-fold decrease in kcat, indicating His-124 to be the general base, unlike MutT, which uses Glu-53 in this role. The H88Q mutant showed a 10(2.3)-fold decrease in kcat, a 4.4-fold increase in K(m)GDPmann, and no change in the pH versus log(kcat) rate profile, indicating an important but unidentified role of His-88 in catalysis. One and two-dimensional NMR studies permitted the sequence specific assignments of the imidazole HdeltaC, H(epsilon)C, N(delta), and N(epsilon) resonances of the four histidines and defined their protonation states. The pK(a) of His-124 (6.94 +/-0.04) in the presence of saturating Mg2+ was comparable to the kinetically determined pK(a) at the same temperature (6.40 +/-0.20). The other three histidines were neutral N(epsilon)H tautomers with pK(a) values below 5.5. Arg-52 and Arg-65 were identified as catalytic residues which interact electrostatically with the GDP leaving group by mutating these residues to Gln and Lys. The R52Q mutant decreased kcat 309-fold and increased K(m)GDPmann 40.6-fold, while the R52K mutant decreased kcat by only 12-fold and increased K(m)GDPmann 81-fold. The partial rescue of kcat, but not of K(m)GDPmann in the R52K mutant, suggests that Arg-52 is a bifunctional hydrogen bond donor to the GDP leaving group in the ground state and a monofunctional hydrogen bond donor in the transition state. Opposite behavior was found with the Arg-65 mutants, suggesting this residue to be a monofunctional hydrogen bond donor to the GDP leaving group in the ground state and a bifunctional hydrogen bond donor in the transition state. From these observations, a mechanism for GDPMH is proposed involving general base catalysis and electrostatic stabilization of the leaving group. << Less

  Biochemistry 41:10834-10848(2002) [PubMed] [EuropePMC]