Kinetic Characterization and Allosteric Inhibition of the <i>Yersinia pestis</i> 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

<div><p>The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from <i>Yersinia pestis</i>. Enzyme assays indicate apparent kinetic constants of K_M^DXP = 252 µM and K_M^NADPH = 13 µM, IC₅₀ values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and K_i values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the <i>Y. pestis</i> MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development.</p></div

Cation specificity of <i>Y. pestis</i> MEP synthase.

<p>Enzyme assays were performed with fixed NADPH (150 µM), DXP (400 µM), and divalent cation (25 mM) concentration. <i>Y. pestis</i> MEP synthase has comparable activity with either Mg²⁺ or Mn²⁺. Assays were performed in duplicate.</p

Dose-response plot of the <i>Y. pestis</i> MEP synthase when preincubated with the inhibitor.

<p>Assays were performed by combining the enzyme with either A) compound <b>15</b> or B) compound <b>16</b> and preincubating at 37°C for 10 min before addition of NADPH and DXP. All assays were performed in duplicate. Activity of the enzyme is relative to an uninhibited control.</p

Purification of recombinant <i>Y. pestis</i> MEP synthase.

<p>A Coomassie stained SDS-PAGE shows two lanes of purified His-tagged MEP synthase alongside a molecular weight marker (MW). His-tagged MEP synthase has a predicted molecular weight of 46.7 kDa. The typical yield of purified protein averaged 30 mg per 1 L shake flask.</p

The MVA and MEP biosynthetic pathways.

<p>A) The MVA pathway is utilized by humans and other eukaryotes, archaebacteria, and certain eubacteria to produce IPP and DMAPP, the building blocks of isoprenoids. The pathway is initiated by the enzymatic condensation of 3 molecules of acetyl-CoA (1) to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) (3), which is then reduced to MVA by HMG-CoA reductase (4) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-M1" target="_blank">[54]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Miziorko1" target="_blank">[55]</a> Subsequent phosphorylation and decarboxylation yield IPP (7) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Amdur1" target="_blank">[56]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-BLOCH1" target="_blank">[57]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-DhePaganon1" target="_blank">[58]</a> which is converted to DMAPP (8) by an isomerase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Agranoff1" target="_blank">[59]</a>. B) The MEP pathway is used by higher plants, the plastids of algae, apicomplexan protozoa, and many eubacteria, including numerous human pathogens. Pyruvate (9) is condensed with glyceraldehyde 3-phosphate (10) to yield 1-deoxy-D-xylulose 5-phosphate (DXP; (11)) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Lange1" target="_blank">[60]</a>, a branch point intermediate with a role in <i>E. coli</i> vitamin B1 and B6 biosynthesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Julliard1" target="_blank">[61]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Julliard2" target="_blank">[62]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Hill1" target="_blank">[63]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Hill2" target="_blank">[64]</a> as well as isoprene biosynthesis. In the first committed step of the <i>E. coli</i> MEP pathway, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (also called MEP synthase, Dxr or IspC) catalyzes the reduction and rearrangement of 11 to yield MEP (12) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-AndrewTKoppisch1" target="_blank">[28]</a>. CDP-ME synthase then converts MEP into 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME; (13)). CDP-ME kinase phosphorylates CDP-ME, which is subsequently cyclized (coupled with the loss of CMP) by cMEPP synthase to yield 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (15) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Rohdich2" target="_blank">[65]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Kuzuyama2" target="_blank">[66]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Kuzuyama3" target="_blank">[67]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Lttgen1" target="_blank">[68]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Herz1" target="_blank">[69]</a>. A reductive ring opening of 15 produces 1-hydroxy-2-methyl-2-butenyl diphosphate (HMBPP; (16)) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Altincicek2" target="_blank">[70]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Campos1" target="_blank">[71]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Hecht1" target="_blank">[72]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Kollas1" target="_blank">[73]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Rohdich3" target="_blank">[74]</a>, which is then reduced to both IPP and DMAPP in a ∼5:1 ratio <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Rohdich1" target="_blank">[8]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Altincicek3" target="_blank">[75]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Cunningham1" target="_blank">[76]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Altincicek4" target="_blank">[77]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-McAteer1" target="_blank">[78]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Rohdich4" target="_blank">[79]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Adam1" target="_blank">[80]</a>. C) The reaction catalyzed by MEP synthase. The intermediate 2-C-methyl-D-erythrose 4-phosphate (18), produced by isomerization via cleavage of the bond between C3 and C4 and formation of a new bond between C2 and C4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Arigoni1" target="_blank">[81]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Putra1" target="_blank">[82]</a>, is subsequently reduced to yield MEP (12).</p

Mechanism of inhibition by e29.

<p>A) Relative to NADPH, e29 is an uncompetitive inhibitor of the purified <i>Y. pestis</i> MEP synthase. B) Relative to DXP, e29 is a noncompetitive inhibitor. C) A model of e29 inhibition. MEP synthase (E) undergoes a conformational change (E*) upon binding of NADPH (N), exposing an allosteric site to which the inhibitor (I) binds. As the inhibitor is noncompetitive with respect to DXP (D), I may bind the E*N or E*ND complex, thereby inhibiting the enzyme.</p

MEP synthase inhibitors.

<p>The structures of fosmidomycin, FR900098, and select rationally designed amide-linked and O-linked inhibitors are shown, including lipophilic prodrug analogs of compound <b>15</b>.</p

Dose-dependent inhibition of the <i>Y. pestis</i> MEP synthase.

<p>IC₅₀ values were determined using A) fosmidomycin or B) FR900098. The R² value for each plot is indicated. Assays were performed in duplicate.</p

Structure–Activity Relationships of the MEPicides: <i>N</i>‑Acyl and <i>O</i>‑Linked Analogs of FR900098 as Inhibitors of Dxr from <i>Mycobacterium tuberculosis</i> and <i>Yersinia pestis</i>

Despite continued research efforts, the threat of drug resistance from a variety of bacteria continues to plague clinical communities. Discovery and validation of novel biochemical targets will facilitate development of new drugs to combat these organisms. The methylerythritol phosphate (MEP) pathway to make isoprene units is a biosynthetic pathway essential to many bacteria. We and others have explored inhibitors of the MEP pathway as novel antibacterial agents. <i>Mycobacterium tuberculosis</i>, the causative agent of tuberculosis, and <i>Yersinia pestis</i>, resulting in the plague or “black death”, both rely on the MEP pathway for isoprene production. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr) catalyzes the first committed step in the MEP pathway. We examined two series of Dxr inhibitors based on the parent structure of the retrohydroxamate natural product FR900098. The compounds contain either an extended <i>N</i>-acyl or <i>O</i>-linked alkyl/aryl group and are designed to act as bisubstrate inhibitors of the enzyme. While nearly all of the compounds inhibited both Mtb and Yp Dxr to some extent, compounds generally displayed more potent inhibition against the Yp homologue, with the best analogs displaying nanomolar IC₅₀ values. In bacterial growth inhibition assays, the phosphonic acids generally resulted in poor antibacterial activity, likely a reflection of inadequate permeability. Accordingly, diethyl and dipivaloyloxymethyl (POM) prodrug esters of these compounds were made. While the added lipophilicity did not enhance <i>Yersinia</i> activity, the compounds showed significantly improved antitubercular activities. The most potent compounds have Mtb MIC values of 3–12 μg/mL. Taken together, we have uncovered two series of analogs that potently inhibit Dxr homologues from Mtb and Yp. These inhibitors of the MEP pathway, termed MEPicides, serve as leads for future analog development

The substrate dependent catalytic activity of <i>Y. pestis</i> MEP synthase.

<p>Shown are the Michaelis-Menten plots of reaction velocity as a function of A) DXP concentration and B) NADPH concentration. Least-squares best fit of the data to the Michaelis-Menten equation produces the kinetic parameters listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone-0106243-t001" target="_blank">Table 1</a>. The R² value for each plot is indicated. All assays were performed in duplicate.</p