Identification in Haloferax volcanii of phosphomevalonate decarboxylase and isopentenyl phosphate kinase as catalysts of the terminal enzyme reactions in an archaeal alternate mevalonate pathway

Abstract

Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase.

FIG 1

Classical and alternate mevalonate pathways for isopentenyl-diphosphate synthesis. In the alternate pathway, PMD corresponds to HVO_1412 and IPK corresponds to HVO_2762.

FIG 2

SDS-PAGE of purified H. volcanii isopentenyl-phosphate kinase and phosphomevalonate decarboxylase. Left lane, molecular weight markers; center lane, phosphomevalonate decarboxylase (4 μg); right lane, isopentenyl-phosphate kinase (4 μg).

FIG 3

IC₅₀ determination for inhibition of HvPMD by 6-fluoromevalonate 5-phosphate (A) and 6-fluoromevalonate 5-diphosphate (B). Differential sensitivity of phosphomevalonate decarboxylase to inhibition by 6-fluoromevalonate 5-phosphate and 6-fluoromevalonate 5-diphosphate is apparent. The curves indicate an IC₅₀ of 16 μM for the monophosphate-containing inhibitor, while the diphosphate-containing compound exhibits no inhibition.

FIG 4

ESI-MS analysis of metabolites of the phosphomevalonate decarboxylase reaction. All spectra were collected in the negative ion mode. (A) MS spectrum of chemically synthesized (R,S)-mevalonate 5-phosphate (m/z = 227). (B) MS spectrum of chemically synthesized isopentenyl 5-phosphate (m/z = 165). (C) MS spectrum of a PMD reaction mixture indicating residual unreacted (S)-mevalonate 5-phosphate (m/z = 227) and showing product isopentenyl 5-phosphate (m/z = 165). (D) MS spectrum of negative control in which no enzyme was included in the reaction mixture. Unreacted (R,S)-mevalonate 5-phosphate substrate is apparent (m/z = 227), but no formation of a product (m/z = 165) peak is observed. (E) MS-MS spectrum of an m/z of 165, yielding phosphate ions (m/z = 79 and m/z = 97).

FIG 5

Proposed H. volcanii phosphomevalonate decarboxylase reaction mechanism. Potential roles of active-site residues serine-105, arginine-142, and aspartate-277 in substrate binding and catalysis are discussed in the text.

FIG 6

Multiple-sequence alignment of phosphomevalonate decarboxylase and mevalonate diphosphate decarboxylases. The sequence of phosphomevalonate decarboxylase from H. volcanii (HvPMD) was aligned with mevalonate diphosphate decarboxylases from Enterococcus faecalis (EfMDD), S. epidermidis (SeMDD), and Homo sapiens (HsMDD). Conserved residues are shaded in black, and similar residues are shaded in gray.