Characterization of Thermophilic Archaeal Isopentenyl Phosphate Kinases

Abstract

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, Keq = 6.3 ± 1. The catalytic efficiencies (V/K) of the proteins were ~2 × 106 M−1s−1. In the reverse direction, ADP was a substrate inhibitor for THA IPK, KiADP = 58 ± 6 µM 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 substrate for the MTH and THA IP kinases. All of the compounds were phosphorylated, although the catalytic efficiency was low for geranyl phosphate.

Identification in Haloferax volcanii of Phosphomevalonate Decarboxylase and Isopentenyl Phosphate Kinase as Catalysts of the Terminal Enzyme Reactions in an Archaeal Alternate Mevalonate Pathway

“…This specific activity represents an ϳ3.5-fold increase in the rate at 4 M KCl versus no added KCl. For isopentenyl phosphate, the K m was 77 Ϯ 9 M, an estimate that falls within the wide range of values (Table 1) reported for IPK from M. jannaschii (5), M. thermautotrophicus, and T. acidophilum (6). The K m for ATP was 290 M, a value Ͼ10-fold larger than that reported for other IPK enzymes from M. thermautotrophicus or T. acidophilum but similar to the report for M. jannaschii.…”

“…(R)-Mevalonate 5-P and (R)-fluoromevalonate monophosphate and diphosphate were synthesized enzymatically, as described by Voynova et al (11). Isopentenyl phosphate was synthesized analogously to the method of Chen and Poulter (6). The purity of the compounds synthesized for this study was Ͼ85%.…”

Expression in Haloferax volcanii of 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Facilitates Isolation and Characterization of the Active Form of a Key Enzyme Required for Polyisoprenoid Cell Membrane Biosynthesis in Halophilic Archaea

“…The results of this study, as well as results of E. coli expression work in Rodwell's lab (7), indicate that HMGCS and reductase function as MVA pathway enzymes, but the latter steps of this pathway are cryptic. It is unclear whether reactions catalyzed by phospho-MVA kinase and MVA 5-diphosphate decarboxylase are critical to isopentenyl 5-diphosphate production or whether this product is produced by a shunt involving a putative MVA 5-phosphate decarboxylase, as well as an isopentenyl 5-phosphate kinase of the type described in other archaea (28,29). Available annotations of Haloferax open reading frames provide no resolution of this ambiguity, since proteins for both metabolic routes are proposed.…”

Characterization of a Feedback-Resistant Mevalonate Kinase from the Archaeon Methanosarcina mazei

“…Putative homologues of all mevalonate pathway genes, excluding the diphosphomevalonate decarboxylase, have been identified in archaea by genomic analysis (3,7,17,19). In addition, putative isopentenyl monophosphate kinases have been identified and characterized from archaea, suggesting the possible utilization of a modified mevalonate pathway for the production of isoprenoids in archaea (8,17) (Fig. 1).…”