Reaction Mechanism of Isopentenyl Phosphate Kinase: A QM/MM Study

Abstract

Isopentenyl phosphate kinase (IPK) catalyzes the Mg-ATP dependent phosphorylation reactions to produce isopentenyl diphosphate, an important precursor in the synthesis of isopentenols. However, the position of the divalent metal ion in the crystal structures of IPK in complex with ATP and its native substrate IP has not been definitively resolved, and as a result ambiguity surrounds the catalytic mechanism of IP, limiting its exploitation as a biofuel and in drug design. Here we report the catalytically competent structure in complex with the metal ion Mg and elucidate the phosphorylation reaction mechanism using molecular dynamic simulations and density functional theory-based quantum mechanics/molecular mechanics calculations (B97d/AMBER99). Comparing the substrate-bound and substrate-free IPK complexes, we observed that substrate binding results in significant conformational change of three residues Lys204, Glu207, and Lys211 located on the αG helix to form a strong salt bridge network with Asp145, which in turn tethers the invariant Ser142 via H-bond interaction. The conformational change shuts the subtrate entrance channel formed between the αG and αE helices. Further, we demonstrate the phosphorylation reaction occurs with a reaction barrier of 17.58 kcal/mol, which is in agreement with the previous experimental kinetic data. We found that a highly conserved Gly8 on a glycine-rich loop, together with Lys14, stabilizes the transition state.

Water-mediated network in the resistance mechanism of fosfomycin

“…7 The corresponding histidine residue His50 in IPK, another kinase in the 'phosphate' subdivision of the AAK family, was also suggested to be crucial for substrate binding and stabilizing the transition state (TS) from previous mutagenesis studies and a QM/MM study. 27,28 In our QM/MM optimized structures of the reactant, transition state and product, His58 remained tethered between the substrate phosphate and the γphosphate of ATP ( Figure 2). Thus our results show that His58 plays a predominant role in positioning the substrate in the catalytic site and stabilizing the transition state during phosphate transfer.…”

“…Starting from the representative structure resulting from the MD simulations of the ATP-FM-Mg 2+ -FomA complex, QM/MM calculations were performed using the QM/MM (B97d/6-31G(d):AMBER99), whereby the QM calculation was performed at the B97d/6-31G(d) theory level, and the MM calculation was carried out using the AMBER99 force field. The B97d functional was chose since it was found to perform well on fellow AAK, IPK 27 . The QM/MM optimized structures of the reactant, TS and product show that Lys18 remains in ionic interactions with Asp208 and the αand γ-phosphate of ATP, helping to position the nucleotide and organize the catalytic site during phosphate transfer (Figure 2c-e).…”

“…This is in good accordance with previous mutagenesis studies on FomA 7 as well as the kinetic and QM/MM studies on its homologous kinase IPK. 27,28 QM/MM studies on IPK 27 revealed that the backbone amide of Gly8 stabilized the TS by forming a hydrogen bond with the β-, γ-bridging oxygen atom of ATP. In the optimized TS structure of FomA, the corresponding glycine residue Gly12 on a γ-turn secondary structure also forms a hydrogen bond with the β-, γ-bridging oxygen of ATP ( Figure S5), indicating Gly12 also participates the stabilization of the TS of FomA.…”

Catalytic mechanism of mevalonate kinase revisited, a QM/MM study

“…Pauling's formula 42 D(n) = D(l) -0.6 log n was used to quantify the associative or dissociative nature of the TS. In the formula the term D(l) is the average length of a P-O bond valued at 1.73 and D(n) is the 22 and PMK 23 , which were found to also follow a direct phosphorylation mechanism with a dissociative nature, as well as Isopentenyl phosphate kinase (IPK) in the mevalonate pathway that belongs to a distinct kinase family, amino acid kinase (AAK) family 43 . Fig.…”

Phosphorylation Mechanism of N-Acetyl-l-glutamate Kinase, a QM/MM Study

“…This highly conserved glycine-rich sequence was also identified to play a crucial role in catalysis of IPK, a homologous AAK kinase whereby highly conserved glycine residues (Gly7 and Gly8) form hydrogen bonds with ATP. 37 to accommodate the entry of substrate and also in a conformation to allow for the product to be released. This is in accordance with E Coli NAGK in absence of substrate and one subunit of NAGK (the subunit A) in complex with the product, both of which exhibit a very open conformation, favourable for the substrate to enter and for the product to release.…”