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1.  Absence of Substrate Channeling between Active Sites in the Agrobacterium tumefaciens IspDF and IspE Enzymes of the Methyl Erythritol Phosphate Pathway† 
Biochemistry  2006;45(11):3548-3553.
The conversion of 2C-methyl-d-erythritol 4-phosphate (MEP) to 2C-methyl-d-erythritol 2,4-cyclodiphosphate (cMEDP) in the MEP entry into the isoprenoid biosynthetic pathway occurs in three consecutive steps catalyzed by the IspD, IspE, and IspF enzymes, respectively. In Agrobacterium tumefaciens the ispD and ispF genes are fused to encode a bifunctional enzyme that catalyzes the first (synthesis of 4-diphosphocytidyl-2-C-methyl d-erythritol) and third (synthesis of 2C-methyl-d-erythritol 2,4-cyclodiphosphate) steps. Sedimentation velocity experiments indicate that the bifunctional IspDF enzyme and the IspE protein associate in solution raising the possibility of substrate channeling among the active sites in these two proteins. Kinetic evidence for substrate channeling was sought by measuring the time courses for product formation during incubations of MEP, CTP, and ATP with the IspDF and IspE proteins with and without an excess of the inactive IspE (D152A) mutant in presence or absence of 30% (v/v) glycerol. The time dependencies indicate that the enzyme-generated intermediates are not transferred from the IspD active site in IspDF to the active site of IspE or from the active site in IspE to the active site in the IspF module of IspDF.
PMCID: PMC2516919  PMID: 16533036
bifunctional; IspDF; IspE; non-channeling
2.  Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase. 
Journal of Bacteriology  1996;178(3):619-624.
Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl diphosphate. Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R. capsulatus. More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabadopsis thaliana, and Clarkia breweri), a worm (Caenorhabiditis elegans), and another eubacterium (Escherichia coli). The R. capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme.
PMCID: PMC177703  PMID: 8550491
3.  Isolation and characterization of isoprene mutants of Escherichia coli. 
Journal of Bacteriology  1989;171(7):3619-3628.
Isoprenoid compounds are found in all organisms. In Escherichia coli the isoprene pathway has three distinct branches: the modification of tRNA; the respiratory quinones ubiquinone and menaquinone; and the dolichols, which are long-chain alcohols involved in cell wall biosynthesis. Very little is known about procaryotic isoprene biosynthesis compared with what is known about eucaryote isoprene biosynthesis. This study approached some of the questions about isoprenoid biosynthesis and regulation in procaryotes by isolating and characterizing mutants in E. coli. Mutants were selected by determining their resistance to low levels of aminoglycoside antibiotics, which require an electron transport chain for uptake into bacterial cells. The mutants were characterized with regard to their phenotypes, map positions, enzymatic activities, and total ubiquinone content. In particular, the enzymes studied were isopentenyldiphosphate delta-isomerase (EC, farnesyldiphosphate synthetase (EC, and higher prenyl transferases.
PMCID: PMC210103  PMID: 2661529
4.  Efficient syntheses of [3-15N]uracil and [3-15N]thymine. 
Nucleic Acids Research  1983;11(18):6497-6504.
Regiospecific syntheses of [3-15N]uracil and [3-15N]thymine are described using [15N]ammonium sulfate as a source of labeled nitrogen. The overall yields are excellent, and the reactions are amenable to production of multigram quantities of labeled material.
PMCID: PMC326389  PMID: 6622258

Results 1-4 (4)