Investigating the reactivities of a polyketide synthase module through fluorescent click chemistry

Article abstract of DOI:10.1039/c3cc47513a

A method for monitoring in vitro polyketide synthesis has been developed whereby nonchromophoric polyketide products are made brightly fluorescent in a simple, rapid, inexpensive, and bioorthogonal manner through CuAAC with a sulforhodamine B azide derivative. the Partner Organisations 2014.

Full text of DOI:10.1039/c3cc47513a

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Investigating the reactivities of a polyketide
synthase module through fluorescent click
chemistry†
Cite this: Chem. Commun., 2014,
50, 5276
Received 1st October 2013,
Accepted 29th October 2013
Amanda Jane Hughes, Matthew R. Tibby, Drew T. Wagner, Johnathan N. Brantley
and Adrian T. Keatinge-Clay*
DOI: 10.1039/c3cc47513a
www.rsc.org/chemcomm
A method for monitoring in vitro polyketide synthesis has been Traditionally, nanogram to microgram quantities of products are
developed whereby nonchromophoric polyketide products are made generated from radiolabeled precursors and visualized by radio-
brightly fluorescent in a simple, rapid, inexpensive, and bioorthogonal TLC.4 While GC/MS has been employed, derivatization is sometimes
manner through CuAAC with a sulforhodamine B azide derivative.
necessary, and the signal-to-noise ratio can be low.5 Previously,
we utilized fluorine NMR to follow fluorinated substrates
Modular polyketide synthases (PKSs) are the largest enzymes known to through reactions catalyzed by overexpressed PKS enzymes in the
man and are responsible for synthesizing some of the most important commonly-employed biocatalytic setting of dialyzed cell lysate;6
human medicines.1,2 While PKSs perform similar chemistry to however, this method requires both sophisticated instrumentation
fatty acid synthases, they possess an assembly-line organization.3 and knowledge of the chemical shifts of generated products. Recent
Sets of enzymes, termed modules, cooperate to extend and advances in biocatalytic methods have aided in scaling up module-
process a growing polyketide chain. These enzymes include a catalyzed reactions – a glucose-fueled NADPH-regeneration system
ketosynthase (KS) that acquires and extends a growing polyketide can provide a constant supply of reduced nicotinamide coenzyme,
chain through a decarboxylative condensation, an acyltransferase and extender units like methylmalonyl-S-N-acetylcysteamine
(AT) that selects the appropriate extender unit for the chain (mm-S-NAC) can be enzymatically generated by MatB for the
extension, an NADPH-dependent ketoreductase (KR) that stereo- price of ATP.7,8 We sought a more general bioorthogonal approach
selectively reduces the KS-generated b-keto group, and an acyl that would allow visualization of polyketide products by HPLC.
carrier protein (ACP) that shuttles the growing polyketide between
Since fluorescent alkyne probes and click chemistry have been used
these enzymes through a thioester linkage to a post-translationally to locate azide-containing glycolipids in the background of a zebrafish,9
appended phosphopantetheinyl arm. The mature polyketide is we reasoned that polyketide products containing a unique chemical
typically released by a C-terminal thioesterase (TE) via cyclization or functionality could be selectively labeled by a complementary fluoro-
hydrolysis. The ability of these enzymes to construct stereochemically- phore in the background of bacterial cell lysate. Here, we showcase a
rich carbon chains containing diverse substituents has inspired quantitative detection method that reports the fate of alkynyl priming
the biosynthetic community to engineer these factories to produce units in module-catalyzed reactions within cell lysate (Fig. 1). Copper(I)-
molecules of medicinal relevance; however, in order to reprogram catalyzed azide alkyne cycloaddition (CuAAC) with the sulforhodamine
PKSs, the reactivities of the enzymes within their modules must B azide derivative enables normally nonchromophoric polyketide
be better understood.
products to be sensitively visualized and identified using fluorescence
There are many challenges associated with investigating the spectroscopy and mass spectrometry.10,11 Employing this method, the
operation of modules, let alone employing them to synthesize
desired compounds: products generated are typically nonchromo-
phoric, substrates such as NADPH and methylmalonyl-CoA are
costly, and modules are often difficult to express and purify.
Department of Chemistry and Biochemistry, The University of Texas at Austin,
1 University Station A5300, Austin, TX, USA. E-mail: adriankc@utexas.edu;
Fax: +1 512 471-8696; Tel: +1 512 471-2977
Fig. 1 Lighting up polyketide products through fluorescent click chemistry.
(A) The sulforhodamine B azide fluorophore. (B) Extender units and terminal
alkyne priming units are incubated with PKS constructs. Reactions are subjected to
click chemistry, and the resulting adducts are visualized by reverse-phase HPLC.
† Electronic supplementary information (ESI) available: Experimental proce-
dures, high-resolution mass spectrum data, low-resolution LC/MS data, and
experiments in buffers containing phosphate and glycerol. See DOI: 10.1039/
c3cc47513a
5276 | Chem. Commun., 2014, 50, 5276--5278
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incorporation of ethanethiol-linked priming and extender units into may not have reacted due to the gradual loss of EryMod6TE activity –
reduced diketide compounds by the terminal module-TE of the precipitate was noticeable in reactions after 6 h.
erythromycin PKS (EryMod6TE) was investigated.
To demonstrate the use of extender unit 2 in the preparative,
All experiments reported here studied the reactivities of biocatalytic syntheses of polyketides, 17 mg of 3 and 13 mg of 2 were
EryMod6TE within E. coli cell lysate. Performing reactions with incubated with EryMod6TE lysate and the NADPH regeneration
polyketide substrates in dialyzed cell lysate saves the time and system. After 16 h, the reaction was acidified, extracted with ethyl
expense associated with protein purification and does not suffer acetate, and chromatographed to afford 3 mg of reduced diketide 5
from appreciable adventitious catalysis.12 EryMod6TE lysate was (22% yield), which was characterized by 1H NMR (ESI†).
incubated with synthetic priming and extender units before
Since the NAC handle of extender unit 1 could be replaced by
initiating CuAAC with the sulforhodamine B azide derivative. ethanethiol, we investigated replacing the NAC handle of priming
Resulting adducts were analyzed by HPLC and characterized by unit 3 with ethanethiol (Fig. 3). Thus, EryMod6TE was incubated
high-resolution mass spectrometry (ESI†). In the experiments with the NADPH regeneration system, extender unit 2, and either
reported here, a fluorimeter was employed for optimal sensitivity, NAC-linked 3 or ethanethiol-linked 9. After 16 h, reactions were
although UV/vis detection also provides an excellent signal.
subjected to CuAAC with sulforhodamine B azide and analyzed by
To decrease costs associated with investigating the reactivities of HPLC. A comparison of the amount of 5* formed from priming
modules and biocatalytically producing polyketides, the use of a units 3 and 9 revealed that EryMod6TE did not significantly
simplified methylmalonyl-CoA analogue as an in vitro extender unit discriminate between NAC or ethanethiol priming unit handles.
was investigated (Fig. 2). Previous in vitro reactions have employed Building on these results, the promiscuity of EryMod6TE towards
enzymatically- or synthetically-generated 1 (mm-S-NAC);8,12 however, NAC- and ethanethiol-linked priming units of varying chain length
the one-step, multigram synthesis of methylmalonyl ethanethioester was assayed. The analysis showed less than 5% difference in product
(mm-S-Et) 2 is much more economical. Thus, EryMod6TE was formation between reduced diketide products 13* and 14*. Thus
incubated with priming unit 3, extender unit 1 or 2, and the NADPH EryMod6TE does not significantly discriminate between priming
regeneration system to produce reduced diketide 5. In all experiments, units based on chain length or handle.
saturating quantities of nicotinamide coenzyme were employed
Having analyzed module-catalyzed reactions employing priming
to obtain optimal activity from EryKR6. After 16 h, reactions units 3 and 6–10, we were interested in probing the reactivity of
were subjected to CuAAC with the sulforhodamine B azide EryMod6TE with a more advanced priming unit, such as b-ketoacyl
derivative and analyzed by HPLC.
ethanethioester 15 (Fig. 4). The chemical synthesis of 15 was performed
The analysis revealed that approximately equal quantities of 5* by C-acylation of 5-hexynoic acid with 2 through a reaction developed
(asterisks denote sulforhodamine adducts) were generated in reac- by Masamune and coworkers.15 Briefly, 2 was reacted with magnesium
tions employing 1 or 2; B35% of the priming unit 3 was accepted, ethoxide to form its magnesium salt, 5-hexynoic acid was reacted with
extended, reduced, and hydrolyzed by EryMod6TE to afford reduced carbonyldiimidazole to form its imidazolide, and these intermediates
diketide 5. Analysis also showed that B50% of priming unit 3 was were reacted with one another. EryMod6TE was incubated for 16 h with
hydrolyzed to carboxylic acid 4 (Fig. 2C and D). TE-catalyzed hydro- priming unit 15, extender unit 2, and the NADPH regeneration system.
lysis of priming units has been previously implicated in the low The reaction was subjected to CuAAC with the sulforhodamine B azide
yields (B10%) of EryMod6TE-catalyzed reactions.13,14 Remaining 3 derivative and analyzed by HPLC. Interestingly, 15 was reduced
Fig. 2 A comparison of in vitro extender units. (A) Synthetic extender units
1 and 2 employed in experiments. (B) Reduced diketide 5 was produced by
EryMod6TE from priming unit 3, 1 or 2, and the NADPH-regeneration
system. EryTE-mediated hydrolysis of 3 yields 4. (C) Fluorimeter-coupled
HPLC analysis of reactions. (D) EryMod6TE employed extender units 1 and
2 interchangeably, as determined through a comparison of the peak areas
of 3*–5* (asterisks indicate sulforhodamine adducts).
Fig. 3 EryKS6 promiscuity towards thiol handles and chain lengths.
(A) Terminal alkyne thioester priming unit substrates. (B) Reduced diketides
5, 13, and 14 were generated from in vitro EryMod6TE reactions with
substrates 3, 6–10. (C) Equivalent product profiles were observed for
NAC-linked priming unit 3 and ethanethiol-linked priming unit 9. (D)
Equivalent product profiles were observed for substrates of varying chain
length, regardless of thiol handle.
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constructs from the pikromycin PKS, greater than 60% yields were
obtained in experiments employing thiophenol-linked priming
units.18 We are currently investigating a panel of alkyne-containing
priming units that display decreased TE-mediated hydrolysis while
remaining substrates for KSs.
The described method above will help answer many funda-
mental questions of single module and bimodular systems
in vitro. Owing to its bioorthogonal nature, it may also be useful
in the quantitation of polyketide intermediates and products
in vivo. The precursor-directed biosynthesis of 15-propargyl erythro-
mycin A is accomplished using a priming unit containing a
terminal alkyne.19 Applying our strategy to such a system would
be very informative, especially for engineered versions of the
erythromycin synthase that do not produce expected products.
In general, we anticipate the described method to greatly
accelerate the investigation and optimization of PKS-catalyzed
biosynthesis of complex polyketide products.
We would like to thank the University of Texas at Austin
mass spectrometry facility for their help in obtaining high-
resolution masses for sulforhodamine B adducts. This research
was supported by the Welch Foundation (F-1712) and the
National Institutes of Health (GM106112). JNB is grateful to
the NSF for support for this research through a pre-doctoral
fellowship.
Fig. 4 EryMod6TE reactivity towards an advanced priming unit. (A) b-Ketoacyl
ethanethioester 15 is reduced by EryKR6. At least one of the resulting stereoisomers
(17) is extended and processed by EryMod6TE to afford ketolactone 19 and
hydroxylactone 20. (B) Reverse-phase HPLC analysis of reactions. Diastereomer(s)
18a are syn and diastereomer(s) 18b are anti, as the retention time of 18a* matches
that of 5*. (C) A bar graph compares the peak areas of the products.
Notes and references
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5278 | Chem. Commun., 2014, 50, 5276--5278
This journal is ©The Royal Society of Chemistry 2014