Identification of an Archaeal type II isopentenyl diphosphate isomerase in methanothermobacter thermautotrophicus.
Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70 degrees C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: K(m), 64 microM; specific activity, 0.476 micromol mg(-1) min(-1); and k(cat), 1.6 s(-1).
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Sam J
SJ
Barkley
Rita M
RM
Cornish
C Dale
CD
Poulter
D000595:Amino Acid Sequence; D019748:Carbon-Carbon Double Bond Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005816:Genetic Complementation Test; D045782:Hemiterpenes; D017013:Methanobacteriaceae / Q000201:enzymology*; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D045782:Hemiterpenes; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase; D008798:Mevalonic Acid