Isoprenoid biosynthesis: Identification and characterization of isopentenyl diphosphate:dimethylallyl diphosphate isomerase from Rhodobacter, characterization of a eukaryotic isopentenyl diphosphate:dimethylallyl diphosphate isomerase and phylogenetic an

The discovery and initial characterization of the first known bacterial IPP:DMAPP isomerase are presented in these studies. A putative Rhodobacter capsulatus gene for IPPIase was identified in the photosynthetic gene cluster by searching protein sequence databases with an amino acid probe based on the active site region of the Schizosaccharomyces pombe IPPIase. The purification and characterization of the cloned gene are reported in these studies. The purified protein was demonstrated to catalyze the conversion of IPP to DMAPP. The Mn++ and Mg++ dependencies were examined in these studies. The R. capsulatus IPPIase was shown to have a higher activity in the presence of Mn++ as compared to Mg++. Furthermore, the k_cat and k_cat/K_m values were 4-fold higher in the Mn ++ containing buffer.; The IPP:DMAPP isomerase from S. pombe was the subject of kinetic studies. The K_M and k_cat values for IPP were determined by standard steady state kinetics which yielded values of 81 μM and 5 s−1 respectively. Product inhibition studies were used to determine the same values for IPP (K_M = 78 μM and k _cat = 4.8 s−1) and also the K_M for DMAPP which was 86 μM. This allowed the calculation of the k_cat value for DMAPP (1.6 s−1) using the Haldane relationship. The Mn++ and Mg++ dependencies were examined in these studies. It was demonstrated Mg++ was the preferred divalent metal ion cofactor for the eukaryotic enzyme.; A phylogenetic analysis of the isoprenyl diphosphate synthase family of proteins allowed for the categorization and description of this family of proteins. Seven groups of phylogenetically distinct enzymes were identified in these studies including bifunctional FPP/GGPP synthases from archeabacteria, FPP synthases from eukaryotes, GGPP synthases from eukaryotes, FPP synthases from bacteria, GGPP synthases from bacteria, chromatoplastic GGPP synthases from plants and long chain synthases. The chromatoplastic synthases from plants were shown to have evolved from a bacterial like FPP synthase. This work represents the first comprehensive classification of the trans isoprenyl diphosphate synthase family of enzymes.