title: Fluorescent substrate analog for monitoring chain elongation by undecaprenyl pyrophosphate synthase in real time.
abstract: Farnesyl pyrophosphate (FPP) is a common substrate for a variety of prenyltransferases for synthesizing isoprenoid compounds. In this study, (2E,6E)-8-O-(N-methyl-2-aminobenzoyl)-3,7-dimethyl-2,6-octandien-1-pyrophosphate (MANT-O-GPP), a fluorescent analog of FPP, was synthesized and demonstrated as a satisfactory substrate for Escherichia coli undecaprenyl pyrophosphate synthase (UPPS) with a K(m) of 1.5 μM and a k(cat) of 1.2s(-1) based on [(14)C]IPP consumption. Interesting, we found that its emission fluorescence intensity at 420 nm increased remarkably during chain elongation, thereby useful for real-time monitoring kinetics of UPPS to yield a K(m) of 1.1 μM and a k(cat) of 1.0 s(-1), consistent with those measured using radiolabeled substrate. Using this assay, the IC(50) of a known UPPS inhibitor farnesyl thiopyrophosphate (FsPP) was confirmed. Our studies provide a convenient and environmentally friendly alternative for kinetics and inhibition studies on UPPS drug target.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, ROC.
authors.forename: Kuo-Hsun
authors.initials: KH
authors.lastname: Teng
authors.forename: Annie P-C
authors.initials: AP
authors.lastname: Chen
authors.forename: Chih-Jung
authors.initials: CJ
authors.lastname: Kuo
authors.forename: Yu-Chin
authors.initials: YC
authors.lastname: Li
authors.forename: Hon-Ge
authors.initials: HG
authors.lastname: Liu
authors.forename: Chao-Tsen
authors.initials: CT
authors.lastname: Chen
authors.forename: Po-Huang
authors.initials: PH
authors.lastname: Liang
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D055162:Biocatalysis; D001671:Biochemistry / Q000379:methods*; D002250:Carbon Radioisotopes; D005456:Fluorescent Dyes / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D011106:Polyisoprenyl Phosphates / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013997:Time Factors; D017186:Titrimetry
chemical_list: D002250:Carbon Radioisotopes; D005456:Fluorescent Dyes; D011106:Polyisoprenyl Phosphates; D012997:Solvents; C502746:farnesyl thiopyrophosphate; C088139:undecaprenyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C023266:undecaprenyl pyrophosphate synthetase