Mevalonate analogues as substrates of enzymes in the isoprenoid biosynthetic pathway of Streptococcus pneumoniae

Abstract

Survival of the human pathogen Streptococcus pneumoniae requires a functional mevalonate pathway, which produces isopentenyl diphosphate, the essential building block of isoprenoids. Flux through this pathway appears to be regulated at the mevalonate kinase (MK) step, which is strongly feedback-inhibited by diphosphomevalonate (DPM), the penultimate compound in the pathway. The human mevalonate pathway is not regulated by DPM, making the bacterial pathway an attractive antibiotic target. Since DPM has poor drug characteristics, being highly charged, we propose to use unphosphorylated, cell-permeable prodrugs based on mevalonate that will be phosphorylated in turn by MK and phosphomevalonate kinase (PMK) to generate the active compound in situ. To test the limits of this approach, we synthesized a series of C 3 -substituted mevalonate analogues to probe the steric and electronic requirements of the MK and PMK active sites. MK and PMK accepted substrates with up to two additional carbons, showing a preference for small substitutents. This result establishes the feasibility of using a prodrug strategy for DPM-based antibiotics in S. pneumoniae and identified several analogues to be tested as inhibitors of MK. Among the substrates accepted by both enzymes were cyclopropyl, vinyl, and ethynyl mevalonate analogues that, when diphosphorylated, might be mechanism-based inactivators of the next enzyme in the pathway, diphosphomevalonate decarboxylase.

Probing Ligand-binding Pockets of the Mevalonate Pathway Enzymes from Streptococcus pneumoniae

“…Products were removed by including PMK and DPM-DC in the reaction mixture to prevent possible product inhibition; therefore, three molar equivalents of NADH are oxidized per MK substrate. (R)-1, -3, -6, -7, and -9 were previously shown to be substrates for MK (9). The concentration of acceptor (Mev or analogue) and MK varied according to the kinetic constants of the acceptor.…”

“…Competent Escherichia coli BL21(DE3) was purchased from Novagen. Racemic mevalonolactone analogues ((R,S)-X), Streptococcus pneumoniae MK, PMK, DPM-DC, and Staphylococcus aureus MK were prepared as described previously (9,11). Chromatography was carried out using an Ä KTA fast-protein liquid chromatography system (Amersham Biosciences).…”

“…Enzymatic Synthesis of Stereochemically Pure Substrates of the Mevalonate Pathway-A series of (R,S)-mevalonolactone analogues ((R,S)-X) were synthesized as racemic mixtures as described (9). Approximately 25 mg of each lactone was treated with five equivalents of KOH at 37°C for 1 h to convert it to the corresponding carboxylate.…”

“…In an effort to enhance the inhibitory properties of Mev⅐pp for use as an antibiotic, we have built a series of ten Mev analogues (9) in which the C 3 -methyl group has been altered to other hydrocarbon substituents ( Table 1). Five of the analogues have linear and branched alkyl groups at C 3 that act as structural probes for the MK allosteric site and the active sites of the three GHMP kinases.…”

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

“…[14][15][16] Isoprenoids have been recognized as anticancer and antimalarial drugs 17,18 as well as potential gasoline replacements. 19,20 Chemical synthesis or modification of isoprenoids is difficult due to their structural complexity, and therefore the large scale of production of isoprenoids in industry is hindered.…”