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

Sean Cahill,Chan Sun Park,Sofia B. Rodriguez,Scott T. Lefurgy,Richard B. Silverman,Thomas S. Leyh

doi:10.1074/jbc.m109.098350

Abstract

Diphosphomevalonate (Mev.pp) is the founding member of a new class of potential antibiotics targeting the Streptococcus pneumoniae mevalonate (Mev) pathway. We have synthesized a series of Mev.pp analogues designed to simultaneously block two steps in this pathway, through allosteric inhibition of mevalonate kinase (MK) and, for five of the analogues, by mechanism-based inactivation of diphosphomevalonate decarboxylase (DPM-DC). The analogue series expands the C(3)-methyl group of Mev.pp with hydrocarbons of varying size, shape, and chemical and physical properties. Previously, we established the feasibility of a prodrug strategy in which unphosphorylated Mev analogues could be enzymatically converted to the active Mev.pp forms by the endogenous MK and phosphomevalonate kinase. We now report the kinetic parameters for the turnover of non-, mono-, and diphosphorylated analogues as substrates and inhibitors of the three mevalonate pathway enzymes. The inhibition of MK by Mev.pp analogues revealed that the allosteric site is selective for compact, electron-rich C(3)-subsitutents. The lack of reactivity of analogues with DPM-DC provided evidence, counter to the existing model, for a decarboxylation transition state that is concerted rather than dissociative. The Mev pathway is composed of three structurally and functionally conserved enzymes that catalyze consecutive steps in a metabolic pathway. The current work reveals that these enzymes exhibit significant differences in specificity toward R-group substitution at C(3) and that these patterns are explained well by changes in the volume of the C(3) R-group-binding pockets of the enzymes.

Highlights

Streptococcus pneumoniae, the primary cause of bacterial meningitis and pneumonia, kills over 4000 people daily worldwide and disproportionately affects children and the elderly [1, 2]
Racemic mixtures of the diphosphorylated forms of these analogues were synthesized chemically starting from the corresponding lactones and used to probe the structural constraints of the allosteric pocket of Mev kinase (MK) and the active site of diphosphomevalonate decarboxylase (DPM-DC)
Twenty-five novel Mev analogues have been tested as substrates and inhibitors of three enzymes that comprise the Mev pathway in S. pneumoniae

Summary

Introduction

Streptococcus pneumoniae, the primary cause of bacterial meningitis and pneumonia, kills over 4000 people daily worldwide and disproportionately affects children and the elderly [1, 2]. The mevalonate (Mev) pathway is essential for the survival of S. pneumoniae in lung and serum [6]. The bacterium uses this pathway to convert Mev to isopentenyl diphosphate, the “building block” of the isoprenoids: a class of 25,000 unique molecules having a wide range of biological functions. We have shown that S. pneumoniae MK is potently (Ki ϭ 500 nM) allosterically inhibited by diphosphomevalonate (Mev1⁄7pp), the third compound in the pathway, whereas the human MK homologue is not [8]. In an effort to enhance the inhibitory properties of Mev1⁄7pp for use as an antibiotic, we have built a series of ten Mev analogues [9] in which the C3-methyl group has been altered to other hydrocarbon substituents (Table 1). The remaining five analogues resemble the alkyl series and have the potential to act as mechanismbased inhibitors of DPM-DC

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Conclusion