Mutation of Archaeal Isopentenyl Phosphate Kinase Highlights Mechanism and Guides Phosphorylation of Additional Isoprenoid Monophosphates

Abstract

The biosynthesis of isopentenyl diphosphate (IPP) from either the mevalonate (MVA) or the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway provides the key metabolite for primary and secondary isoprenoid biosynthesis. Isoprenoid metabolism plays crucial roles in membrane stability, steroid biosynthesis, vitamin production, protein localization, defense and communication, photoprotection, sugar transport, and glycoprotein biosynthesis. Recently, an alternative branch of the MVA pathway was discovered in the archaeon Methanocaldococcus jannaschii involving a small molecule kinase, isopentenyl phosphate kinase (IPK). IPK belongs to the amino acid kinase (AAK) superfamily. In vitro, IPK phosphorylates isopentenyl monophosphate (IP) in an ATP and Mg2+-dependent reaction producing IPP. Here, we describe crystal structures of IPK from M. jannaschii refined to nominal resolutions of 2.0−2.8 Å. Notably, an active site histidine residue (His60) forms a hydrogen bond with the terminal phosphate of both substrate and product. This His residue serves as a marker for a subset of the AAK family that catalyzes phosphorylation of phosphate or phosphonate functional groups; the larger family includes carboxyl-directed kinases, which lack this active site residue. Using steady-state kinetic analysis of H60A, H60N, and H60Q mutants, the protonated form of the Nε2 nitrogen of His60 was shown to be essential for catalysis, most likely through hydrogen bond stabilization of the transition state accompanying transphosphorylation. Moreover, the structures served as the starting point for the engineering of IPK mutants capable of the chemoenzymatic synthesis of longer chain isoprenoid diphosphates from monophosphate precursors.

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

“…The catalytic rate constant (k cat ) of 16.3/s was comparable to several other values reported for these enzymes. There is considerable discrepancy between the kinetic constants reported for M. jannaschii IPK by two different laboratories (5,14). Differences in the assay buffer concentration, pH, and temperature (or other unspecified assay methodology) may partially account for these discrepancies.…”

Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis

“…(17) identified an enzyme from Methanocaldococcus jannaschii capable of phosphorylating isopentenyl phosphate ( 9 ) to IPP ( 1 ). A modified MVA pathway was thus proposed (Figure 1B), in which mevalonate-5-phosphate ( 7 ) is decarboxylated to 9 and then phosphorylated by isopentenyl phosphate kinase (IPK) to form IPP ( 1 ) (18–20). However, the proposed phosphomevalonate decarboxylase (PMD, 7 → 9 conversion) has yet to be identified.…”

Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants

“…1). Instead of PMK, archaea possess isopentenyl phosphate kinase (IPK), catalyzing the conversion of isopentenyl phosphate (IP) to IPP (8,9). The discovery of Significance Terpenoids, one of the largest and most diverse classes of natural products, are found in all living organisms, where they play essential roles in growth and development, respiration and photosynthesis, and interactions with the environment.…”