A Single Amino Acid Mutation Converts (R)-5-Diphosphomevalonate Decarboxylase into a Kinase

Abstract

The biosynthesis of isopentenyl diphosphate, a fundamental precursor for isoprenoids, via the mevalonate pathway is completed by diphosphomevalonate decarboxylase. This enzyme catalyzes the formation of isopentenyl diphosphate through the ATP-dependent phosphorylation of the 3-hydroxyl group of ()-5-diphosphomevalonate followed by decarboxylation coupled with the elimination of the 3-phosphate group. In this reaction, a conserved aspartate residue has been proposed to be involved in the phosphorylation step as the general base catalyst that abstracts a proton from the 3-hydroxyl group. In this study, the catalytic mechanism of this rare type of decarboxylase is re-investigated by structural and mutagenic studies on the enzyme from a thermoacidophilic archaeon The crystal structures of the archaeal enzyme in complex with ()-5-diphosphomevalonate and adenosine 5'--(3-thio)triphosphate or with ()-5-diphosphomevalonate and ADP are newly solved, and theoretical analysis based on the structure suggests the inability of proton abstraction by the conserved aspartate residue, Asp-281. Site-directed mutagenesis on Asp-281 creates mutants that only show diphosphomevalonate 3-kinase activity, demonstrating that the residue is required in the process of phosphate elimination/decarboxylation, rather than in the preceding phosphorylation step. These results enable discussion of the catalytic roles of the aspartate residue and provide clear proof of the involvement of a long predicted intermediate, ()-3-phospho-5-diphosphomevalonate, in the reaction of the enzyme.

Conversion of Mevalonate 3-Kinase into 5-Phosphomevalonate 3-Kinase by Single Amino Acid Mutations

“…4A and B). The chemical shift of the signal was in good agreement with the reported chemical shift of the C-3 signal of MVA-3,5-BP, the product of M3P5K from T. acidophilum (10), and with that of MVA-3-P-5-PP, which was produced by SsoDMD mutants that lacked decarboxylation activity (16). Moreover, the signal seemed to acquire an additional hyperfine coupling that resulted from the two-bond 13 C-31 P coupling, which was obvious in the C-3 signals of MVA-3,5-BP and MVA-3-P-5-PP (10,16).…”

“…The chemical shift of the signal was in good agreement with the reported chemical shift of the C-3 signal of MVA-3,5-BP, the product of M3P5K from T. acidophilum (10), and with that of MVA-3-P-5-PP, which was produced by SsoDMD mutants that lacked decarboxylation activity (16). Moreover, the signal seemed to acquire an additional hyperfine coupling that resulted from the two-bond 13 C-31 P coupling, which was obvious in the C-3 signals of MVA-3,5-BP and MVA-3-P-5-PP (10,16). Slight but obvious changes between the spectra of the samples reacted with or without the E140S mutant were also found in the vicinity of the signals of the other For the data shown in panels A and C, the sample from the negative-control reaction with water was used; for the data shown in panels B and D, the sample from the reaction with the E140S mutant was used.…”

“…In their study, the absence of the decarboxylase activity in M3K was in part attributed to the absence of an aspartate residue conserved in DMD and PMD, but they did not test their hypothesis by mutagenesis. In our recent work, the Asp281 residue of DMD from a thermophilic archaeon, Sulfolobus solfataricus, was replaced with, for example, its counterpart in M3K, threonine (16). As a result, the M3K-mimicking mutant still recognized MVA-5-PP as the substrate, but the decarboxylase activity was lost, while the 3-kinase activity that yields 3-phospho-5-diphosphomevalonate (MVA-3-P-5-PP) was retained.…”

Modified mevalonate pathway of the archaeon Aeropyrum pernix proceeds via trans -anhydromevalonate 5-phosphate

“…The C 5 compound IPP is synthesized by the decarboxylation of MVA5PP accompanied by a detachment of its 3-hydroxyl group. To catalyze the reaction, diphosphomevalonate decarboxylase (DMD) consumes ATP to temporarily phosphorylate MVA5PP and form mevalonate 3-phosphate 5-diphosphate inside its catalytic pocket as shown recently by our mutagenic study (3). Detachment of the 3-phosphate group of the intermediate triggers decarboxylation to yield IPP.…”

Visualizing the enzyme mechanism of mevalonate diphosphate decarboxylase

“…The γ-phosphate group of ATP is transferred to MVAPP to make the 3′-phosphate-MVAPP intermediate, which is then subjected to dephosphorylation and decarboxylation to produce IPP. Although it was proposed that the ATP-dependent decarboxylation of MVAPP 26 might be initiated via deprotonation of 3′-OH of MVAPP by a conserved aspartic acid (D282 in MDD from Enterococcus faecalis, and D283 in MDD from Staphylococcus epidermidis), followed by the transfer of the γ-phosphate of ATP to MVAPP 27 , recent mutagenesis studies on MDD from Sulfolobus solfataricus (MDD SS ) have demonstrated that the catalytic Asp residue may function in dephosphorylation and decarboxylation of the 3′-phosphate-MVAPP intermediate, rather than deprotonation of the 3′-OH group of MVAPP 28 . However, the phosphoryl transfer efficiencies of the D281T or D281V mutants of MDD SS are lower than the wild-type MDD SS , implying some involvement of the Asp in this step.…”