Kinetic characterization and allosteric inhibition of the Yersinia pestis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase)

• Published in: PloS one 2014-08, Vol.9 (8), p.e106243-e106243
• Main Authors: Haymond, Amanda, Johny, Chinchu, Dowdy, Tyrone, Schweibenz, Brandon, Villarroel, Karen, Young, Richard, Mantooth, Clark J, Patel, Trishal, Bases, Jessica, San Jose, Geraldine, Jackson, Emily R, Dowd, Cynthia S, Couch, Robin D
• Format: Article
• Language: English
• Subjects: Aldose-Ketose Isomerases - chemistry; Aldose-Ketose Isomerases - genetics; Allosteric properties; Allosteric Regulation; Analogs; Antibiotic resistance; Antibiotics; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Binding sites; Biochemistry; Biology and Life Sciences; Biosynthesis; Catalysis; Chemistry; Clinical trials; D-Xylulose 5-phosphate; Drug development; E coli; Enzymes; Epidemics; Erythritol - analogs & derivatives; Erythritol - biosynthesis; Erythritol - chemistry; Escherichia coli; Fosfomycin - analogs & derivatives; Fosfomycin - chemistry; Fosmidomycin; High-throughput screening; Identification; Isomerization; Kinases; Kinetics; Medical screening; Medicine and Health Sciences; NADP; Natural products; Phosphates; Proteins; Reductoisomerase; Signal transduction; Synechocystis; Terpenes; Ubiquinone; Xylulose; Yersinia pestis; Yersinia pestis - enzymology; Yersinia pestis - genetics
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0106243

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 Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KMDXP = 252 µM and KMNADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis 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.