ESCHERICHIA-COLI IMIDAZOLEGLYCEROL PHOSPHATE DEHYDRATASE - SPECTROSCOPIC CHARACTERIZATION OF THE ENZYMATIC PRODUCT AND THE STERIC COURSE OFTHE REACTION

Recombinant strains of Escherichia coli have been developed for the hi gh-level production of imidazoleglycerol phosphate dehydratase (IGPD) and imidazoleacetol phosphate aminotransferase (IAP aminotransferase). These protein sources facilitated the determination of the IGPD react ion stereochemistry and enabled the development of a continuous spectr ophotometric enzyme assay for the IGPD reaction. D-erythro-IGP and D-e rythro-[3-H-2]IGP were generated using a chemoenzymatic approach. D-(( -))-[3-H-2]Ribose5-phosphate was prepared synthetically, starting from diacetone-D-glucose, and converted enzymatically to D-erythro-[3-H-2] IGP. In separate reactions, D-erythro-IGP and D-erythro-[3-H-2]IGP wer e converted to IAP using E. coli IGPD, The resulting IAP was transform ed directly to histidinol using the coupled activities of E, coli IAP aminotransferase and histidinol phosphate phosphatase. The enzymatical ly generated histidinol samples were analyzed by H-1 and 2H NMR and co mpared to a synthetically prepared sample of (2S,3S*)-[3-H-2]histidin ol. This analysis demonstrated that the E. coil IGPD reaction proceeds with inversion of configuration at C-3, and the proton added to C-3 o f IAP during the course of the dehydration is derived from the solvent . The observed stereochemical outcome is consistent with the idea that if the IGPD reaction proceeds through an enol intermediate, then taut omerization of the enol to IAP must be enzyme-mediated, The product of the IGPD reaction, IAP, has been characterized by NMR spectroscopy in aqueous solution. IAP undergoes rapid exchange of the C-3 protons wit h the bulk medium and exists as a mixture of the ketone and its hydrat e (a geminal diol). Additional solution chemistry of IAP was observed using UV-vis and EPR spectroscopy and is consistent with the idea that IAP coordinates to Mn2+ in a bi- or tridentate fashion in aqueous sol utions.