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 Mg(2+)-dependent reaction producing IPP. Here, we describe crystal structures of IPK from M. jannaschii refined to nominal resolutions of 2.0-2.8 A. 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 Nepsilon(2) 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.

Figure 1

The amino acid kinase (AAK) family members. Isopentenyl phosphate kinase (IPK) reaction depicted across the top. Representative family members displayed from left to right: carbamate kinase (CK), aspartokinase (AK), glutamate-5-kinase (G5K), N-acetyl-l-glutamate kinase (NAGK), fosfomycin resistance kinase (FomA), and uridine monophosphate kinase (UMPK). The percent sequence identities relative to IPK are listed above each enzyme. Reactions shaded green utilize a phosphate or phosphonate phosphoryl acceptor, while the reactions shaded red utilize carbamate or carboxylate groups as phosphate acceptors.

Figure 2

Primary sequence, tertiary architecture, and active site snapshots of IPK. a) Primary sequence of IPK from M. jannaschii aligned with E. coli NAGK. The color coding of each motif correlates with its color shown on the three-dimensional model. b) Global view of the IPK dimer (top) and a close-up view of the dimerization interface (bottom). Motifs positioned near the dimerization interface are gray (or pink) for one monomer and black (or red) for the other. c) Ribbon diagram of the IPK monomer. The structure is colored using a blue to red gradient from the N- to C-terminus. The C-terminal ATP-binding domain contains a β-sulfate residing in a location coinciding with the β-phosphate of ATP. The ATP analog AMPPNP is faintly colored and blended into the background (modeled from PDBID: 1gs5) and serves as a reference for the putative location of ATP in IPK. The crystallographically observed isopentenyl phosphate (IP) substrate is shown bound within the N-terminal domain. d) The active sites of IPK complexed with IP (left), IPP (middle), and IPPβS (right). Electron density surrounding each ligand (dark and light blue are contoured to 1σ and 0.6σ, respectively) shown as 2Fo−Fc omit electron density maps, where the ligands were removed before a round of refinement and subsequent phase and map calculations.

Figure 3

Comparative close-up views of the nucleotide phosphate-binding region of the IPK and FomA active sites. a) Monomer A of the IPK−IPPβS complex depicting the β-sulfate ion and the surrounding residues. b) Monomer B of the IPK−IPPβS complex oriented as in panel a. c) FomA complexed with the ATP analog AMPPNP and fosfomycin (PDB ID: 3d41) (15). As depicted here, the β-sulfate ion in both IPK monomers shares a similar position and interacts with the same residues as does the β-phosphate group on AMPPNP in FomA.

Figure 4

a) Close-up view of the N-terminal domain depicting the isopentenyl tail and the surrounding hydrophobic residues. The motifs surrounding the active site are colored as follows: β2−αB glycine-rich loop (red), αB helix (magenta), β3−β4 hairpin (yellow), β4−αC loop (green), N-terminal portion of the αC helix (cyan), and the β9−β10 hairpin (blue). Residues within van der Waals contact of the isopentenyl chain include Ile86, Met90, and Ile156. b) Dual conformation of the β1−αA loop in monomer A of the IPK−IP complex. One conformation places the loop close to the β2−αB loop and the IP substrate, while the other conformation places the loop in close proximity to the β-sulfate ion.

Figure 5

a) Tertiary structure superposition of monomers A and B of the IPK−IP complex. The rmsd between the two monomers is 1.31 Å. b−c) Close-up views of residues proximal to and hydrogen bonding with the α-phosphate of IP in monomers A (panel b) and B (panel c). In monomer B, a water molecule bridges the side-chain amino group of Lys6 and a nonbridging oxygen atom of the IP phosphate. d) Tertiary structure superposition of monomers A and B of the IPK−IPP complex. The rmsd between the two monomers is 1.39 Å. e−f) Views of the multiple conformers of IPP (labeled as IPP-a and IPP-b) in both monomers A (panel e) and B (panel f).

Figure 6

Farnesyl phosphate (FP) phosphorylation by IPK chain length mutants. a) The coupled IPK−sesquiterpene synthase reaction used to test for FP transphosphorylation. b) Comparative bar graph depicting several IPK tunnel mutants qualitatively tested for their ability to convert FP to FPP (expressed as a percentage of maximal production of 5-epi-aristolocene produced from IPK-generated FPP using wild-type IPK and identical concentrations of wild-type tobacco 5-epi-aristolochene synthase incubated for equivalent lengths of time).