Monitoring biocatalytic transformations mediated by polyketide synthase enzymes in cell lysate via fluorine NMR

Article abstract of DOI:10.1055/s-0032-1316554

The biocatalytic employment of modular polyketide synthase enzymes in cell lysate has become a viable route to preparative quantities of synthetically valuable polyketide fragments. We report the quantitative, uninvasive, and continuous monitoring of such biocatalytic reactions by observing trifluoromethyl-bearing substrates via ¹⁹F NMR spectroscopic analysis. To demonstrate the utility of this technique, we followed reactions catalyzed by a thioesterase and several ketoreductases.

Full text of DOI:10.1055/s-0032-1316554

1840▌
LETTER
lMetteor nitoring Biocatalytic Transformations Mediated by Polyketide Synthase
Enzymes in Cell Lysate via Fluorine NMR
Monitoring Biocatalysis in Lysate by Fluorine NMR
Shawn K. Piasecki,a Adrian T. Keatinge-Clay*a,b
a
Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A5300, Austin, TX 78712, USA
b
Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300, Austin, TX 78712, USA
Fax +1(512)4718696; E-mail: adriankc@mail.utexas.edu
Received: 12.04.2012; Accepted after revision: 25.05.2012
fluorine into any of its biomolecules (few organisms do).3
Abstract: The biocatalytic employment of modular polyketide syn-
Furthermore, the 19F nucleus is (i) 83% as intrinsically
thase enzymes in cell lysate has become a viable route to prepara-
sensitive as 1H, (ii) 100% naturally abundant, (iii) isoster-
tive quantities of synthetically valuable polyketide fragments. We
report the quantitative, uninvasive, and continuous monitoring of
ic with hydrogen, and (iv) very responsive to its electronic
such biocatalytic reactions by observing trifluoromethyl-bearing environment.4 These properties have been exploited in
substrates via 19F NMR spectroscopic analysis. To demonstrate the
utility of this technique, we followed reactions catalyzed by a
thioesterase and several ketoreductases.
drug discovery efforts; one technique, called FABS
(fluorine atoms for biochemical screening), identifies in-
hibitors by detecting a decrease in the rate of conversion
from a fluorinated substrate into its product by 19F NMR
Key words: biosynthesis, biocatalysis, polyketide synthase en-
zymes, polyketide, fluorine NMR
spectroscopic analysis.5
First, we observed a hydrolysis reaction mediated by the
erythromycin thioesterase (EryTE), which is known to
Modular polyketide synthases (PKSs) are enzymatic as-
catalyze the hydrolysis of acyl thioesters.2b,6 We sought to
sembly lines that produce complex polyketides such as the
determine how accurately the kinetics of EryTE-mediated
antibacterial erythromycin and the antifungal amphoteri-
hydrolysis could be measured within the cell lysate by 19F
cin.1 Employing the catalytic power of PKS enzymes to
NMR spectroscopic analysis compared to the more tradi-
generate polyketides possessing desired substituents and
tional technique of employing HPLC and a UV detector.
stereochemistries has long been a goal in biosynthetic en-
Thus,
3,3,3-trifluoropropionyl-S-N-acetylcysteamine
gineering. Recently, advances in employing isolated PKS
enzymes as biocatalysts have enabled access to prepara-
tive quantities of polyketide fragments.2 These biocatalyt-
ic reactions are performed in the cell lysate of the
overexpression host (e.g., Escherichia coli), thus maxi-
mizing the quantity of enzyme and avoiding resource-
intensive protein purification. Because cell lysate is a
complex mixture of all the soluble biomolecules produced
by the overexpression host, other fates are possible for a
substrate entered into such a system in addition to the de-
sired transformation. To evaluate such biocatalytic reac-
tions we sought a quantitative and uninvasive technique
that could continuously monitor both substrates and prod-
ucts.
(NAC; 1) was incubated in EryTE-containing cell lysate
both in an NMR tube and in a separate vessel so that for
every spectrum acquired, a sample was also quenched for
later HPLC analysis. 19F NMR spectroscopic analysis
(without proton decoupling) yielded a triplet for each spe-
cies due to splitting of the fluorine resonance by the adja-
cent methylene hydrogens. The triplet of substrate 1
appeared at δ = –63.00 ppm, while the 3,3,3-trifluoropro-
pionate product (2) showed triplets at δ = –63.55 and
–63.70 ppm, possibly due to two different interactions
with counterions (Figure 1). Kinetic characterization was
performed by measuring the change in concentration of 1
by both 19F NMR spectroscopic analysis and reversed-
phase HPLC. The determined kcat and Km parameters
agreed within the error limits. Thus, 19F NMR spectro-
scopic analysis accurately measured the kinetics of EryTE
on 1 in cell lysate (kcat = 0.077 ± 0.010 s–1, Km = 39.3 ±
4.5 mM, kcat/Km = 1.97 ± 0.14 M–1s–1). Previous kinetic
analysis of EryTE towards similar, unfluorinated thioester
substrate analogs gave comparable values.6 Kinetic anal-
ysis is not only more facile by 19F NMR spectroscopic
analysis than by HPLC, but also more informative — gen-
eration of the nonchromophoric product 2 was observed in
the EryTE reaction.
To these ends, NMR spectroscopy would be useful; how-
ever, observing the conversion of substrate into product
via 1H NMR spectroscopic analysis is complicated by the
high background generated by the hydrogen-containing
components of biocatalytic reactions (e.g., biomolecules,
co-substrates, buffering agent, glycerol, DMSO) and by
significant noise from water even when suppression meth-
ods are employed. However, if the substrates contained a
trifluoromethyl group and were observed by 19F NMR
spectroscopic analysis, no background would interfere
with measurements because E. coli does not incorporate
We next sought to monitor a more advanced biocatalytic
transformation driven by an NADPH-regeneration system
comprised of glucose dehydrogenase, NADP+, and
glucose.2a,7 Thus, the conversion of 3-oxo-5,5,5-trifluoro-
pentanoyl-S-NAC (3) into (3R)-hydroxy-5,5,5-trifluoro-
SYNLETT 2012, 23, 1840–1842
Advanced online publication: 04.07.2012
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9
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1
4
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DOI: 10.1055/s-0032-1316554; Art ID: ST-2012-Y0317-L
© Georg Thieme Verlag Stuttgart · New York

LETTER
Monitoring Biocatalysis in Lysate by Fluorine NMR
1841
pentanoyl-S-NAC (4) by the KR from the first module of ferences generated at the α- and β-carbon atoms by stereo-
the tylosin PKS (TylKR1) was observed by 19F NMR controlled, KR-mediated reductions. Thus, 2-methyl-3-
spectroscopic analysis (Figure 2). The triplet from sub- oxo-5,5,5-trifluoropentanoyl-S-NAC (5) was incubated
strate 3 appeared at δ = –63.18 ppm, while the triplet from with each of the four KRs: AmpKR2 (the KR from the
product 4 (confirmed by mass spectrometry and NMR second module of the amphotericin PKS), RifKR7 (the
analysis) appeared at δ = –63.60 ppm. The rate of the re- KR from the seventh module of the rifamycin PKS),
action measured by 19F NMR spectroscopic analysis con- TylKR1, and EryKR1 (the KR from the first module of
curred with the rate measured by HPLC (at 30 mM of 3, the erythromycin PKS), which are known to set (2R,3S),
Vo was calculated to be 0.20 ± 0.02 mM·min–1 by 19F (2S,3S), (2R,3R), and (2S,3R) stereochemistries, respec-
NMR spectroscopic analysis and 0.23 ± 0.05 mM·min–1 tively, when reducing 2-methyl-3-oxopentanoyl-S-NAC
by HPLC), approximately matching the rate anticipated (Figure 3).2a,7,8 The triplet for substrate 5 appeared at δ =
from previous kinetic studies of TylKR1 on (2RS)-meth- –63.23 ppm. Notably, the triplets for the syn- and anti-
yl-3-oxopentanoyl-S-NAC (0.81 mM·min–1).7a
products were well-resolved (0.34 ppm); the triplet for the
syn- products 6 and 9 appeared at δ = –63.68 ppm, and
the triplet for anti- products 7 and 8 appeared at δ =
–63.34 ppm. That the diastereomers could be readily dis-
tinguished within cell lysate highlights the sensitivity of
this technique.
Figure 2 Monitoring biocatalysis mediated by TylKR1 in cell lysate
Figure 3 19F NMR resolution of diastereomers generated by stereo-
controlled, KR-mediated biocatalysis in cell lysate
Figure 1 Accurate kinetic characterization of EryTE-mediated hy-
drolysis of fluorinated 1 in cell lysate via 19F NMR spectroscopic
analysis
Whether the trifluoromethyl-bearing substrate analogs
would be processed in the same manner as the nonfluor-
inated substrates normally entered into these biocatalytic
reactions was initially unclear; however, EryTE and the
KRs were shown to catalyze the desired transformations,
stereoselectively in the case of the KRs.2a Studies report-
We then sought to determine whether the terminal fluor-
ine atoms in an ε-trifluoro, α-substituted β-ketothioester
were sensitive enough to distinguish stereochemical dif-
© Georg Thieme Verlag Stuttgart · New York
Synlett 2012, 23, 1840–1842

1842
S. K. Piasecki, A. T. Keatinge-Clay
LETTER
Spangenberg and the UT Austin NMR core facility for help collec-
ting kinetic data.
ing the acceptance of monofluorinated thioesters by the
erythromycin PKS and the studies presented here suggest
that PKS enzymes are generally tolerant towards fluori-
nated substrates and that biocatalytic syntheses of
polyketides can be reliably evaluated by 19F NMR spec-
troscopic analysis.9 The ability to follow nonchromophor-
ic substrates by 19F NMR spectroscopic analysis enables
monitoring of substrates and products in biocatalytic reac-
tions generating reduced polyketides such as triketide lac-
tone chiral building blocks. The rapid detection of
thioester hydrolysis or other undesired transformations
will facilitate the optimization of biocatalytic reactions
performed in cell lysate.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.SnuIpofoiprmntgirStanoIuifpog
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Acknowledgment
This research was funded by the Robert A. Welch Foundation
(Grant F-1712). We thank Joshua Detelich and Madeline Detelich
for help synthesizing fluorinated substrates. We also thank Angela
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Synlett 2012, 23, 1840–1842
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