2
54 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Brise n˜ o-Roa et al.
nor KHCO (PTE cofactors) were added when PTE-wt and PTE-
3
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h5 were assayed because no enhancement in activity was observed
in titration trials (data not shown). Whenever mentioned, DMF was
present at a final concentration of 1% v/v. The hydrolysis rates of
the fluorogenic NA analogues in the absence of enzyme varied
according to their structure, but even for the least stable substrate,
(
10
was still ∼10 lower than the rate catalyzed by PTE (Supporting
Information).
The emission and excitation spectra were measured in 50 mM
HEPES at pH 8.0. The emission spectrum was obtained with an
excitation at 350 nm. Conversely, the excitation spectrum was
obtained by reading fluorescence emitted at 460 nm. Under these
conditions, the maximal excitation and emission wavelength were
(
17) Gopal, S.; Rastogi, V.; Ashman, W.; Mulbry, W. Mutagenesis of
organophosphorus hydrolase to enhance hydrolysis of the nerve agent
VX. Biochem. Biophys. Res. Commun. 2000, 279, 516-519.
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375 and 465 nm, respectively. The quantum yield (Φ) of the
(
19) Chen-Goodspeed, M.; Sogorb, M. A.; Wu, F.; Raushel, F. M.
Enhancement, relaxation, and reversal of the stereoselectivity for
phosphotriesterase by rational evolution of active site residues.
Biochemistry 2001, 40, 1332-1339.
fluorogenic leaving group, 3-chloro-7-hydroxy-4-methylcoumarin,
was obtained by the comparative method of Williams et al.,58 taking
as standard 7-diethylamino-4-methylcoumarin (Sigma Chemicals
Ltd, Dorset, UK) for which the quantum yield is known (Φ )
(20) Li, W.; Lum, K. T.; Chen-Goodspeed, M.; Sogorb, M. A.; Raushel,
F. M. Stereoselective detoxification of chiral sarin and soman
analogues by phosphotriesterase. Bioorg. Med. Chem. 2001, 9, 2083-
0
0
.73).59 The Φ value for 7-hydroxy-3-chloro-4-methylcoumarin was
.67.
2091.
(
21) Hill, C. M.; Li, W.-S.; Thoden, J. B.; Holden, H. M.; Raushel, F. M.
Enhanced degradation of chemical warfare agents through molecular
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Acknowledgment. The authors thank Leonid Gaidukov and
Dan Tawfik for providing the mammalian paraoxonases and
for their suggestions and discussion. We acknowledge the UK
Ministry of Defence and the Medical Research Council UK for
funding this work. Luis Brise n˜ o-Roa was supported by a Gates
Cambridge Scholarship and an Overseas Research Student
Award.
(22) Griffiths, A. D.; Tawfik, D. S. Directed evolution of an extremely
fast phosphotriesterase by in vitro compartmentalization. EMBO J.
2003, 22, 24-35.
(23) Aharoni, A.; Griffiths, A. D.; Tawfik, D. S. High-throughput screens
and selections of enzyme encoding genes. Curr. Opin. Chem. Biol.
2005, 2, 210-6.
Supporting Information Available: Additional synthetic and
NMR data for organophosphorus intermediates; LC-MS data for
the fluorogenic analogues and their hydrolysis rates in the absence
of enzyme. This material is available free of charge via the Internet
at http://pubs.acs.org.
(24) Harel, M.; Aharoni, A.; Gaidukov, L.; Brumshtein, B.; Khersonsky,
O. et al. Structure and evolution of the serum paraoxonase family of
detoxifying and anti-atherosclerotic enzymes. Nat. Struct. Mol. Biol.
2004, 11, 412-419.
(
(
(
(
25) Aharoni, A.; Gaidukov, L.; Yagur, S.; Toker, L.; Silman, I. et al.
Directed evolution of mammalian paraoxonases PON1 and PON3
for bacterial expression and catalytic specialization. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 482-487.
26) Bernath, K.; Hai, M.; Mastrobattista, E.; Griffiths, A. D.; Magdassi,
S. et al. In vitro compartmentalization by double emulsions: sorting
and gene enrichment by fluorescence activated cell sorting. Anal.
Biochem. 2004, 325, 151-157.
27) Aharoni, A.; Bernath, K.; Magdassi, S.; Tawfik, D. S. Directed
evolution of thiolactonases by single-cell phenotyping and fluorescent
activated sorting of double emulsion compartments. ACS Chem. Biol.,
in press.
28) Mastrobattista, E.; Tally, V.; Chanudet, E.; Kelly, B. T.; Griffiths,
A. D. Discovering novel evolutionary pathways to â-galactosidases
using in vitro compartmentalisation and fluorescence activated sorting
of double emulsions. ACS Chem. Biol., in press.
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