title: Stereoselective incorporation of an unsaturated isoleucine analogue into a protein expressed in E. coli.
abstract: The unsaturated amino acid 2-amino-3-methyl-4-pentenoic acid (E-Ile) was prepared in the form of its (2S,3S),(2R,3R) and (2S,3R),(2R,3S) stereoisomeric pairs. The translational activities of SS-E-Ile and SR-E-Ile were assessed in an E. coli strain rendered auxotrophic for isoleucine. SS-E-Ile was incorporated into the test protein mouse dihydrofolate reductase (mDHFR) in place of isoleucine at a rate of up to 72 %; SR-E-Ile yielded no conclusive evidence for incorporation. ATP/PPi exchange assays indicated that SS-E-Ile was activated by the isoleucyl-tRNA synthetase at a rate comparable to that characteristic of isoleucine; SR-E-Ile was activated approximately 100-times more slowly than SS-E-Ile.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, MC 210-41, Pasadena, California 91125, USA.
authors.forename: Marissa L
authors.initials: ML
authors.lastname: Mock
authors.forename: Thierry
authors.initials: T
authors.lastname: Michon
authors.forename: Jan C M
authors.initials: JC
authors.lastname: van Hest
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D007532:Isoleucine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D007533:Isoleucine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods / Q000592:standards; D051379:Mice; D008968:Molecular Conformation; D010421:Pentanoic Acids / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
chemical_list: C509180:2-amino-3-methyl-4-pentenoic acid; D010421:Pentanoic Acids; D007532:Isoleucine; D013762:Tetrahydrofolate Dehydrogenase; D007533:Isoleucine-tRNA Ligase