Escherichia coli open reading frame 696 Is idi, a nonessential gene encoding isopentenyl diphosphate isomerase

Isopentenyl diphosphate isomerase catalyzes the interconversion of isopente nyl 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 reac tions. In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pat hway. 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 fo r enzymes in the isoprenoid pathway. E. coli FH12 was constructed by disrup tion of the chromosomal idi gene with the aminoglycoside 3'-phosphotransfer ase 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 plasmi d grew on minimal medium, thereby establishing that idi is a nonessential g ene. Although the V-max of the bacterial protein was 20-fold lower than tha t of its yeast counterpart, the catalytic efficiencies of the two enzymes w ere similar through a counterbalance in K(m)s. The E. coli protein requires Mg2+ or Mn2+ for activity. The enzyme contains conserved cysteine and glut amate active-site residues found in other IPP isomerases.