PMID:10419945
| Citation |
Hahn, FM, Hurlburt, AP and Poulter, CD (1999) Escherichia coli open reading frame 696 is idi, a nonessential gene encoding isopentenyl diphosphate isomerase. J. Bacteriol. 181:4499-504 |
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| Abstract |
Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the E. coli chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase, idi, is not clustered with other known genes for enzymes in the isoprenoid pathway. E. coli FH12 was constructed by disruption of the chromosomal idi gene with the aminoglycoside 3'-phosphotransferase gene and complemented by the wild-type idi gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44 degrees C and FH12 lacking the plasmid grew on minimal medium, thereby establishing that idi is a nonessential gene. Although the V(max) of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance in K(m)s. The E. coli protein requires Mg(2+) or Mn(2+) for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases. |
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| Keywords |
Carbon-Carbon Double Bond Isomerases/genetics; Carbon-Carbon Double Bond Isomerases/isolation & purification; Carbon-Carbon Double Bond Isomerases/metabolism; Chromosome Mapping; Chromosomes, Bacterial; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/growth & development; Genes, Bacterial; Kinetics; Open Reading Frames; Polymerase Chain Reaction; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Restriction Mapping |
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