title: Kinetic characterization and allosteric inhibition of the Yersinia pestis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase).
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Amanda
authors.initials: A
authors.lastname: Haymond
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Chinchu
authors.initials: C
authors.lastname: Johny
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Tyrone
authors.initials: T
authors.lastname: Dowdy
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Brandon
authors.initials: B
authors.lastname: Schweibenz
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Karen
authors.initials: K
authors.lastname: Villarroel
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Richard
authors.initials: R
authors.lastname: Young
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Clark J
authors.initials: CJ
authors.lastname: Mantooth
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Trishal
authors.initials: T
authors.lastname: Patel
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Jessica
authors.initials: J
authors.lastname: Bases
authors.affiliation: Department of Chemistry, George Washington University, Washington DC, United States of America.
authors.forename: Geraldine
authors.initials: G
authors.lastname: San Jose
authors.affiliation: Department of Chemistry, George Washington University, Washington DC, United States of America.
authors.forename: Emily R
authors.initials: ER
authors.lastname: Jackson
authors.affiliation: Department of Chemistry, George Washington University, Washington DC, United States of America.
authors.forename: Cynthia S
authors.initials: CS
authors.lastname: Dowd
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Robin D
authors.initials: RD
authors.lastname: Couch
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics; D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D004896:Erythritol / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry; D005578:Fosfomycin / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D015010:Yersinia pestis / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005578:Fosfomycin; C024640:fosmidomycin; C024639:3-(N-acetyl-N-hydroxy)aminopropylphosphonic acid; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D019747:Aldose-Ketose Isomerases; D004896:Erythritol