Phosphorylation Mechanism of Phosphomevalonate Kinase: Implications for Rational Engineering of Isoprenoid Biosynthetic Pathway Enzymes

Abstract

Mevalonate pathway is of important clinical, pharmaceutical and biotechnological relevance.However, lack of the understanding of the phosphorylation mechanism of the kinases in this pathway has limited rationally engineering the kinases in industry. Here the phosphorylation reaction mechanism of a representative kinase in the mevalonate pathway, phosphomevalonate kinase, was studied by using molecular dynamics and hybrid QM/MM methods. We find that a conserved residue (Ser106) is reorientated to anchor ATP via a stable H-bond interaction. In addition, Ser213 located on the α-helix at the catalytic site is repositioned to further approach the substrate, facilitating the proton transfer during the phosphorylation. Furthermore, we elucidate that Lys101 functions to neutralize the negative charge developed at the β-, γ-bridging oxygen atom of ATP during phosphoryl transfer. We demonstrate that the dissociative catalytic reaction occurs via a direct phosphorylation pathway. This is the first study on the phosphorylation mechanism of a mevalonate pathway kinase. The elucidation of the catalytic mechanism not only sheds light on the common catalytic mechanism of GHMP kinase superfamily, but also provides the structural basis for engineering the mevalonate pathway kinases to further exploit their applications in the production of a wide range of fine chemicals such as biofuels or pharmaceuticals.3

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

“…Previously the catalytic mechanism of PMK, a GHMP kinase in the mevalonate pathway, was revealed using a combined QM/MM study. 5 It was found that reaction occurred via a direct phosphorylation mechanism and a positively charged residue, Lys101, stabilizes the negative charge at the cleaved β-,γ-bridging oxygen of ATP.…”

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

“…Arg241 plays a pivotal role in organizing the highly flexible triphosphate tail of ATP, as well as neutralizing the negative charge accumulated at the β-,γ-bridging oxygen of ATP during phosphate transfer. Thus far, MVK, GALK 22 and PMK 23 have all been shown through QM/MM studies to have an inline, direct phosphate transfer mechanism instead of a catalytic base mechanism. Homoserine kinase, which lacks such potential catalytic base residue in the active site would also function through direct phosphorylation.…”

“…Recently, QM/MM studies on GHMP kinases galactokinase (GALK) 22 and phosphomevalonate kinase (PMK) 23 revealed that these enzymes actually function through a direct phosphorylation mechanism. Thus it is necessary to resolve the uncertainty around the mechanism of MVK, a representative GHMP kinase that was conventionally deems to catalyse the phosphorylation via the catalytic base mechanism.…”

“…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.…”

“…it was suggested that Asp190 in the active site of GALK2 is too far from the substrate to function as a catalytic base 21 . Recently, QM/MM studies revealed that the reactions catalyzed by GALK and PMK occur via direct phosphorylation mechanism [22][23] .…”