Kinetic characterization and phosphoregulation of the Francisella tularensis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase).
Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (K(M)(DXP) = 104 microM, K(M)(NADPH) = 13 microM, k(cat)(DXP) = 2 s(-1), k(cat)(NADPH) = 1.3 s(-1)), an IC(50) for fosmidomycin of 247 nM, and a K(i) for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg(+2) as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.
Jawaid
Safdar
S
Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
Seidle
Heather
H
Zhou
Weidong
W
Abdirahman
Hafsa
H
Abadeer
Maher
M
Hix
Joseph H
JH
van Hoek
Monique L
ML
Couch
Robin D
RD
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000890:Anti-Infective Agents / Q000494:pharmacology; D002070:Butadienes / Q000737:chemistry; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D005578:Fosfomycin / Q000031:analogs & derivatives / Q000494:pharmacology; D005603:Francisella / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D045782:Hemiterpenes / Q000096:biosynthesis / Q000737:chemistry; D057166:High-Throughput Screening Assays; D007700:Kinetics; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010420:Pentanes / Q000737:chemistry; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification; D040681:Structural Homology, Protein; D013379:Substrate Specificity / Q000187:drug effects
D000890:Anti-Infective Agents; D002070:Butadienes; D002413:Cations, Divalent; D045782:Hemiterpenes; D009097:Multienzyme Complexes; D010420:Pentanes; D011994:Recombinant Proteins; C005059:isoprene; D005578:Fosfomycin; C024640:fosmidomycin; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D019747:Aldose-Ketose Isomerases