Investigation of the active sites of 1-aminocyclopropane-1-carboxylic acid oxidase and the pep-utilizing enzymes 3-deoxy-D-arabino-heptulosonate 7-phosphate and 3-deoxy-D-manno-octulosonate 8-phosphate synthases.

The enzymes Escherichia coli DAH7P synthase (phe) and E. coli KDO8P synthase catalyze an unusual condensation between phosphoenolpyruvate (PEP) and a phosphorylated monosaccharide, D-erythrose 4-phosphate (E4P) and D-arabinose 5-phosphate (ASP), respectively. Both enzymatic reactions occur via a nucleophilic attack at the re-face of the carbonyl of the phosphorylated monosaccharide by the si-face of C3PEP even though sequence homology studies indicate sparse sequence homology between both enzymes (18% sequence identity). The catalytic mechanisms and the active site topologies of E. coli DAH7P synthase (phe) and E. coli KDO8P synthase were investigated using site-directed mutagenesis, chemical modification studies, and substrate analogues. Both E. coli KDO8P synthase and E. coli DAH7P synthase (phe) may share a common active site topology as demonstrated utilizing analogues of the respective phosphorylated monosaccharide substrate. The stereochemistry at C2 of the phosphorylated monosaccharide influenced the Km (binding affinity) for both E. coli DAH7P synthase and E. coli KDO8P synthase. Sequence alignment studies between recombinant metallo-KDO8P synthases and a DAH7P synthase cloned from Thermotoga maritima indicate that both enzymes may share a common metal binding motif. This finding provides additional evidence supporting a common active site topology may exist between DAH7P synthases and KDO8P synthases. Both KDO8P synthase and DAH7P synthase (phe) may share a similar mechanism. Site-directed mutagenesis and chemical modification studies of DAH7P synthase (phe) demonstrate that DAH7P synthase (phe) has one essential histidine residue, H268, which is homologous to the residue, H202, in E. coli KDO8P synthase. These two histidine residues occupy analogous positions in the active sites of both enzymes implying that both residues may perform the same role. In addition, studies with substrate analogues of either ASP or E4P which are one-carbon shorter, either E4P for KDO8P synthase or D,L-glyceraldehyde 3-phosphate for DAH7P synthase, demonstrate that both enzymatic reactions may be initiated by a nucleophilic attack of water at C2PEP.PhDBiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127767/2/3029351.pd