X-ray structures of isopentenyl phosphate kinase

Abstract

Isoprenoid compounds are ubiquitous in nature, participating in important biological phenomena such as signal transduction, aerobic cellular respiration, photosynthesis, insect communication, and many others. They are derived from the 5-carbon isoprenoid substrates isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In Archaea and Eukarya, these building blocks are synthesized via the mevalonate pathway. However, the genes required to convert mevalonate phosphate (MP) to IPP are missing in several species of Archaea. An enzyme with isopentenyl phosphate kinase (IPK) activity was recently discovered in Methanocaldococcus jannaschii (MJ), suggesting a departure from the classical sequence of converting MP to IPP. We have determined the high-resolution crystal structures of isopentenyl phosphate kinases in complex with both substrates and products from Thermoplasma acidophilum (THA), as well as the IPK from Methanothermobacter thermautotrophicus (MTH), by means of single-wavelength anomalous diffraction (SAD) and molecular replacement. A histidine residue (His50) in THA IPK makes a hydrogen bond with the terminal phosphates of IP and IPP, poising these molecules for phosphoryl transfer through an in-line geometry. Moreover, a lysine residue (Lys14) makes hydrogen bonds with nonbridging oxygen atoms at P(alpha) and P(gamma) and with the P(beta)-P(gamma) bridging oxygen atom in ATP. These interactions suggest a transition-state-stabilizing role for this residue. Lys14 is a part of a newly discovered "lysine triangle" catalytic motif in IPKs that also includes Lys5 and Lys205. Moreover, His50, Lys5, Lys14, and Lys205 are conserved in all IPKs and can therefore serve as fingerprints for identifying new homologues.

Figure 1

The alternate route in the MVA pathway. In Archaea, the last two steps in the formation of IPP are reversed. Phosphomevalonate is decarboxylated to form IP, which is then phosphorylated to give IPP.

Figure 2

Tertiary structure of THA IPK. (a) A ribbon diagram of the THA IPK monomer with the helices and strands labeled. The region that connects β14 and αG is disordered in the crystal and not modeled in the structure. (b) The THA IPK primary sequence and secondary structures in the same color scheme as in (a). The residues are numbered below the primary sequence. The segment between β14 and αG (which contains αF) is not modeled.

Figure 3

Homodimers of THA and MTH IPK’s. (a) The THA IPK homodimer, with secondary structures involved in the dimer interface labeled. (b) The MTH IPK homodimer. The dyadic axis is perpendicular to the core β-sheet that runs across the dimer and projects toward the viewer.

Figure 4

IP and IPP binding sites (a) The IP binding site showing the amino acid residues that form the hydrophobic interior of the pocket and limit the chain length specificity of the enzyme. Hydrogen bonds between active site residues and the phosphate moiety of IP are shown as broken yellow lines. A water molecule in the active site is modeled as a red sphere, acting as a hydrogen bonding bridge between IP and Asp144. (b) The IPP binding site.

Figure 5

Stereo view comparisons of THA IPK and NAGK active sites. THA IPK (green) and E. coli NAGK (red) align 207 Cα with an r.m.s.d. of 3.0 Å. Sequence identity is 14%. The bound molecules of THA IPK (IP and ATP) and NAGK (NAG and AMPPNP) align well in the active site. Overlapping active site residues between THA IPK and NAGK are also labeled. Some residues in the IP and NAG binding sites are also modeled, showing residues that give rise to the specificity of these two enzymes.

Figure 6

Proposed catalytic mechanism for IPK. Catalytic residues and selected hydrogen bonds are shown. The “missing” Mg²⁺ ion is positioned based on overlap with the structure of FomA.

Figure 7

Electron density maps of the active sites of THA and MTH IPK’s contoured at 1 RMSD. (a) The THA IPK active site with bound substrates, IP and ATP. (b) The THA IPK active site with bound products, IPP and ADP. The Gly8 residue is obscured by P_γ of ATP. (c) The MTH IPK active site with bound glycerol and ADP, showing the putative catalytic residues, Gly8 and Ser9. Putative catalytic residues are labeled and water molecules are shown as red spheres. Ligand omit maps for IP and ATP, and IPP and ADP, can be found in Supplementary Figure 2, panels a and b.