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

摘要

Recombinant strains of Escherichia coli have been developed for the high-level production of imidazoleglycerol phosphate dehydratase (IGPD) and imidazoleacetol phosphate aminotransferase (IAP aminotransferase). These protein sources facilitated the determination of the IGPD reaction stereochemistry and enabled the development of a continuous spectrophotometric enzyme assay for the IGPD reaction. D-erythro-IGP and D-erythro-[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 were converted to IAP using E. coli IGPD, The resulting IAP was transformed directly to histidinol using the coupled activities of E, coli IAP aminotransferase and histidinol phosphate phosphatase. The enzymatically generated histidinol samples were analyzed by H-1 and 2H NMR and compared to a synthetically prepared sample of (2S*,3S*)-[3-H-2]histidinol. This analysis demonstrated that the E. coil IGPD reaction proceeds with inversion of configuration at C-3, and the proton added to C-3 of 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 tautomerization 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 with the bulk medium and exists as a mixture of the ketone and its hydrate (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 solutions. [References: 57]