Probing ligand-binding pockets of the mevalonate pathway enzymes from Streptococcus pneumoniae

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.

FIGURE 1.

The mevalonate pathway. The conversion of mevalonate to isopentenyl diphosphate occurs in three ATP-dependent steps catalyzed by GHMP family kinases: MK, mevalonate kinase; PMK, phosphomevalonate kinase; DPM-DC, diphosphomevalonate decarboxylase.

FIGURE 2.

DPM-DC inactivation hypothesis. The dissociative model of Mev·pp decarboxylation (shown for the 9·pp analogue) begins with phosphorylation of the C₃-OH by ATP. Phosphate then ionizes, leaving a C₃ carbocation, which either rapidly decarboxylates to form the double-bonded product (left resonance form) or, in the case of analogues 6·pp through 10·pp, rearranges and is quenched by a nucleophile on the protein surface (right resonance form), forming a covalent adduct. Opening the cyclopropyl ring of the 9·pp analogue results in a homoallyl carbocation that is stabilized, relative to the ring-intact carbocation, as a result of the release of the ∼27 kcal/mol ring strain (29, 30).

FIGURE 3.

Structural determinants of substrate selectivity in the mevalonate pathway. Models of the Mev pathway enzymes are shown with their respective acceptor substrates bound to the active site. Surfaces (light blue) represent the van der Waals contact surface of the protein model. Substrates Mev, Mev·p, and Mev·pp are shown in stick representation and colored by atom type: C in cyan, H in white, O in red, and P in orange. The C₃-methyl group is shown with hydrogens attached. A, S. pneumoniae MK (magenta, PDB: 2OI2) bound to Mev. The Mev C₃-methyl group points toward His-20, which forms a hydrogen-bonded network (green dashes) with a water molecule (green sphere), Gly-256, and Val-23. B, S. pneumoniae PMK (green, PDB: 3GON) with Mev·p. The Mev·p C₃-methyl group points toward a hydrophobic network capped by Tyr-19. C, homology model of S. pneumoniae DPM-DC based on the S. pyogenes DPM-DC (white, PDB: 2GS8) with Mev·pp manually positioned (see “Experimental Procedures”). The Mev·pp C₃-methyl points into a large water-filled cavity composed of nine conserved residues. Images were generated by using PyMOL (19).

FIGURE 4.

NMR determination of the fate of the cyclopropyl ring. A, partial one-dimensional ¹H NMR spectra at 600 MHz obtained at various times during the enzymatic reaction of 9·pp with DPM-DC. A spectrum was taken every 7 min after the addition of enzyme. The time stamp of a given reaction time is shown. The multiplets at 0.40 ppm/0.35 ppm and at 0.69 ppm/0.50 ppm represent the cyclopropyl methylene protons of the starting material and product, respectively. The integrated areas of the multiplets are preserved during the reaction indicating that all starting material is converted to product. B, expansion of two-dimensional ¹H-¹³C HSQC with multiplicity editing of the product after enzymatic reaction of 9·pp. The signals at 0.69 ppm (¹H)/5.5 ppm (¹³C) and at 0.50 ppm (¹H)/5.5 ppm (¹³C) are from the methylene protons of the cyclopropyl ring. These chemical shifts are characteristic of a cyclopropyl ring and demonstrate that the ring is preserved during the enzymatic reaction.