Characterization of thermophilic archaeal isopentenyl phosphate kinases.
Archaea synthesize isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the essential building blocks of isoprenoid compounds, from mevalonate (MVA). However, an analysis of the genomes of several members of the Archaea failed to identify genes for the enzymes required to convert phosphomevalonate (PM) to IPP in eukaryotes. The recent discovery of an isopentenyl kinase (IPK) in Methanocaldococcus jannaschii (MJ) suggests a new variation of the MVA pathway where PM is decarboxylated to give isopentenyl phosphate (IP), which is phosphorylated to produce IPP. A blast search using the MJ protein as a probe revealed a subfamily of amino acid kinases that include the fosfomycin resistance protein fomA, which deactivates the antibiotic by phosphorylation of its phosphonate residue in a reaction similar to the conversion of IP to IPP. IPK genes were cloned from two organisms identified in the search, Methanothermobacter thermautotrophicus (MTH) and Thermoplasma acidophilum (THA), and the His-tagged recombinant proteins were purified by Ni-NTA chromatography. The enzymes catalyze the reversible phosphorylation of IP by ATP, K(eq) = 6.3 +/- 1. The catalytic efficiencies (V/K) of the proteins were approximately 2 x 10(6) M(-1) s(-1). In the reverse direction, ADP was a substrate inhibitor for THA IPK, K(i)(ADP) = 58 +/- 6 microM, but not for MTH IPK. Both enzymes were active over a broad range of pH and temperature. Five compounds, dimethylallyl phosphate, isopentenyl thiolophosphate, 1-butyl phosphate, 3-buten-1-yl phosphate, and geranyl phosphate, were evaluated as alternative substrates for the MTH and THA IP kinases. All of the compounds were phosphorylated, although the catalytic efficiency was low for geranyl phosphate.
Chen
Mo
M
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Poulter
C Dale
CD
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045782:Hemiterpenes / Q000096:biosynthesis / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017013:Methanobacteriaceae / Q000201:enzymology* / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry; D010766:Phosphorylation; D010802:Phylogeny; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013822:Thermoplasma / Q000201:enzymology* / Q000378:metabolism
D019843:Archaeal Proteins; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C004809:isopentenyl pyrophosphate; D011494:Protein Kinases; C402428:isopentenyl monophosphate kinase