ribulose-5-phosphate and 3-hydroxybutanal

3-Keto-l-gulonate 6-phosphate decarboxylase (KGPDC) and d-arabino-hex-3-ulose 6-phosphate synthase (HPS) are members of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily [Wise, E., Yew, W. S., Babbitt, P. C., Gerlt, J. A., and Rayment, I. (2002) Biochemistry 41, 3861-3869], a group of homologous enzymes that share the (beta/alpha)(8)-barrel fold. KGPDC catalyzes a Mg(2+)-dependent decarboxylation reaction in the catabolic pathway of l-ascorbate utilization by Escherichia coli K-12 [Yew, W. S., and Gerlt, J. A. (2002) J.Bacteriol. 184, 302-306]; HPS catalyzes a Mg(2+)-dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate in formaldehyde-fixing methylotrophic bacteria [Kato, N., Ohashi, H., Hori, T., Tani, Y., and Ogata, K. (1977) Agric. Biol. Chem. 41, 1133-1140]. Our previous studies of the KGPDC from E. coli established the occurrence of a stabilized cis-enediolate intermediate [Yew, W. S., Wise, E., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 43, 6427-6437; Wise, E., Yew, W. S., Gerlt, J. A., and Rayment, I. (2004) Biochemistry 43, 6438-6446]. Although the mechanism of the HPS-catalyzed reaction has not yet been investigated, it also is expected to involve a Mg(2+)-stabilized cis-enediolate intermediate. We now have discovered that the KGPDC from E. coli and the HPS from Methylomonas aminofaciens are both naturally promiscuous for the reaction catalyzed by the homologue. On the basis of the alignment of the sequences of orthologous KGPDC's and HPS's, four conserved active site residues in the KGPDC from E. coli were mutated to those conserved in HPS's (E112D/R139V/T169A/R192A): the value of the k(cat) for the promiscuous HPS activity was increased as much as 170-fold (for the E112D/R139V/T169A/R192A mutant), and the value of k(cat)/K(m) was increased as much as 260-fold (for the E112D/R139V/T169A mutant); in both cases, the values of the kinetic constants for the natural KGPDC activity were decreased. Together with the structures of mutants reported in the accompanying manuscript [Wise, E. L., Yew, W. S., Akana, J., Gerlt, J. A., and Rayment, I., accompanying manuscript], these studies illustrate that large changes in catalytic efficiency can be accomplished with only modest changes in active site structure. Thus, the (beta/alpha)(8)-barrel fold shared by members of the OMPDC suprafamily appears well-suited for the evolution of new functions.

Topics: Alanine; Aldehyde-Lyases; Aldehydes; Amino Acid Substitution; Arginine; Aspartic Acid; Carboxy-Lyases; Catalysis; Decarboxylation; Enzyme Stability; Escherichia coli Proteins; Evolution, Molecular; Formaldehyde; Glutamic Acid; Histidine; Ketoses; Methylomonas; Orotidine-5'-Phosphate Decarboxylase; Ribulosephosphates; Stereoisomerism; Threonine; Valine

On the basis of (13)C and deuterium isotope effects, L-ribulose-5-phosphate 4-epimerase catalyzes the epimerization of L-ribulose 5-phosphate to D-xylulose 5-phosphate by an aldol cleavage to the enediolate of dihydroxyacetone and glycolaldehyde phosphate, followed by rotation of the aldehyde group and condensation to the epimer at C-4. With the wild-type enzyme, (13)C isotope effects were 1.85% at C-3 and 1.5% at C-4 at pH 7, with the values increasing to 2.53 and 2.05% at pH 5.5, respectively. H97N and Y229F mutants at pH 7 gave values of 3.25 and 2.53% at C-3 and 2. 69 and 1.99% at C-4, respectively. Secondary deuterium isotope effects at C-3 were 2.5% at pH 7 and 3.1% at pH 5.5 with the wild-type enzyme, and 4.1% at pH 7 with H97N. At C-4, the corresponding values were 9.6, 14, and 19%. These data suggest that H97N shows no commitments, while the wild-type enzyme has an external commitment of approximately 1.4 at pH 7 and an internal commitment independent of pH of approximately 0.6. The Y229 mutant shows only the internal commitment of 0.6. The sequence of the epimerase is similar to those of L-fuculose-1-phosphate and L-rhamnulose-1-phosphate aldolases for residues in the active site of L-fuculose-1-phosphate aldolase, suggesting that Asp76, His95, His97, and His171 of the epimerase may be metal ion ligands, and Ser44, Gly45, Ser74, and Ser75 may form a phosphate binding pocket. The pH profile of V/K for L-ribulose 5-phosphate is bell-shaped with pK values of 5.94 and 8.24. The CD spectra of L-ribulose 5-phosphate and D-xylulose 5-phosphate differ sufficiently that the epimerization reaction can be followed at 300 nm.

Topics: Acetaldehyde; Aldehyde-Lyases; Aldehydes; Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Carbohydrate Epimerases; Carbon Isotopes; Catalysis; Circular Dichroism; Deuterium; Dihydroxyacetone; Escherichia coli; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Mutation; Pentosephosphates; Ribulosephosphates; Sequence Alignment; Stereoisomerism; Thermodynamics