In vivo reporter labeling of proteins via metabolic delivery of coenzyme A analogues

Article abstract of DOI:10.1021/ja052911k

We demonstrate site-specific reporter labeling of proteins within live cells. By accessing the coenzyme A (CoA) metabolic pathway with a cell-permeable pantetheine analogue, we deliver CoA-bound reporter molecules for post-translational protein modificati

Full text of DOI:10.1021/ja052911k

Published on Web 07/23/2005
In Vivo Reporter Labeling of Proteins via Metabolic Delivery of Coenzyme A
Analogues
Kristine M. Clarke, Andrew C. Mercer, James J. La Clair, and Michael D. Burkart*
Department of Chemistry and Biochemistry, UniVersity of California, San Diego, 9500 Gilman DriVe, La Jolla,
California 92093-0358
Received May 18, 2005; E-mail: mburkart@ucsd.edu
Selective chemical control of biochemical processes within a
living cell enables the study and modification of natural biological
systems in ways that may not be obtained through in vitro
experiments.1a,b Accordingly, access to promiscuous metabolic
pathways has provided a unique chemical entry into small molecule
engineering in vivo.1c We recently introduced a method for covalent
reporter labeling of carrier proteins using promiscuous phospho-
pantetheinyltransferase (PPTase) enzymes and reporter-labeled
coenzyme A (CoA).2a To date, this method has been limited to in
vitro and cell-surface protein labeling,2,3 as CoA derivatives have
not been shown to penetrate the cell. To overcome this obstacle,
we demonstrate addition of labeled metabolic precursors to cell
culture that results in cellular uptake and metabolic conversion into
active, labeled CoA derivatives. We now establish a chemoenzy-
matic route to protein modification via a four-step enzymatic
sequence in vivo.
The chemical synthesis and activity of CoA has been studied
for well over a half-century, yet the full biosynthesis of the cofactor
has only recently been elucidated in prokaryotes and eukaryotes.4,5
CoA is biosynthesized by five enzymes in Escherichia coli, CoAA-
CoAE, while eukaryotes contain a fusion of CoAD and CoAE,
PPAT/DPCK. Knowledge of the substrate specificity of these
enzymes remains incomplete, although some evidence points to
promiscuity within this pathway. Early studies on CoAA indicated
that the enzyme will also accept pantetheine as a substrate.6 This
ability has since been used in the chemoenzymatic synthesis of
CoA analogues.7 This permissiveness suggests using the CoA
biosynthetic pathway to convert reporter-labeled pantetheine to
reporter-labeled CoA in vivo. To this end, the synthesis of
Figure 1. In vivo metabolic labeling of a carrier protein (CP) via cellular
uptake of pantetheine analogue 1 and conversion to CoA analogue 4 by
CoAA, CoAD, and CoAE. This process is followed by reaction of a PPTase
fluorescently labeled pantetheine analogues provides a direct link
to reporter-labeled post-translational modifications.8
A key facet to any in vivo modification relies on access to a
viable uptake mechanism. We chose the nonhydrolyzable, fluores-
cent pantetheine analogue 1 in an effort to probe its uptake and
retention. In E. coli, excess pantetheine is exported from the cell,
and a sodium-dependent symporter has been suggested to import
pantetheine from the surrounding media.9 Once inside the cell,
pantetheine analogue 1 can be phosphorylated by CoAA and ATP
to yield 2 (Figure 1). Further processing by CoAD and CoAE should
proceed by stepwise adenylation of 2 to dephospho-CoA 3 and
further phosphorylation to yield CoA analogue 4. This analogue
would now be captured within the cell and, therefore, remain viable
for downstream processing events. Given the correct strain, this
process could be used to selectively transfer the label in 4 onto a
carrier protein domain (5).
with 4 and a carrier protein yielding labeled protein 5. VibB, a natural fusion,
is depicted as a carrier protein fused with isochorismate lyase (ICL).
Scheme 1. Synthesis of Pantetheine Mimic 1
In selecting analogue 1, we chose to eliminate the thioester
linkage found in natural CoA metabolites, thereby avoiding potential
side reactions due to hydrolysis and oxidation in vivo. To create
the non-natural connectivity, we began by protecting the 1,3-diol
of pantothenic acid (7) as p-methoxybenzylacetal 8 (Scheme 1).
Acetal 8 was coupled to fluorescent amine 6,10 providing the
protected pantetheine mimic 9 in 77% yield. Final deprotection
under acidic conditions provided analogue 1 in an overall yield of
37% from 6 and 7.
To test if analogue 1 would be an acceptable substrate for E.
coli CoAA, CoAD, and CoAE, we reconstituted the CoA pathway
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J. AM. CHEM. SOC. 2005, 127, 11234-11235
10.1021/ja052911k CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
transformed E. coli with 1 mM 1 and IPTG, lysate from the isolated
cells was visualized via fluorescent SDS-PAGE (Figure 3b),
indicating post-translational modification of VibB by Sfp and
fluorescent CoA analogue 4. Fluorescent intensity reaches a
maximum at 5-8 h post-induction (Figure 3b). Lower intensity is
also readily visualized when incubation is carried out in 100 µM 1
(Figure 3c). As a control, no labeling was seen from addition of 1
to cell lysate from induced, untreated cells.
The flux of pantetheine through E. coli is high due to constituent
biosynthesis and rapid export.9 We anticipate that other organisms
that do not produce pantetheine precursors or lack export mecha-
nisms may be effective hosts for this protein-labeling technique.
The fact that 1 was viable in E. coli provides strong support for
the general application of these tools to natural product pathway
elucidation, fusion protein localization, and the analysis of complex
expression patterns. The tolerance to modification throughout this
pathway should allow delivery of a wide variety of chemical motifs
to in vivo processes. We are currently investigating the use of these
methods to visualize and manipulate heterologous proteins, fusion
constructs, and natively expressed carrier protein domains.
Figure 2. In vitro enzymatic reconstitution of the metabolic-labeling
process. (a) HPLC analysis of the stepwise conversion of 1 to fluorescent
CoA analogue 4. (b) Fluorescent SDS-PAGE gel depicting the labeling
of VibB by 4 with Sfp. Note intermediates 2 and 3 can also be visualized
through gel analysis.
Acknowledgment. The authors thank Dr. Debbie Tahmassebi
at the University of San Diego for use of instrumentation. Funding
was provided by the University of California, San Diego, Depart-
ment of Chemistry and Biochemistry, NSF CAREER award under
Grant No. 0347681, and UCSD Center for AIDS Research. K.C.
was supported by a US Department of Education GAANN
fellowship.
Supporting Information Available: Experimental procedures,
NMR data for compounds 1 and 6-9. This material is available free
of charge via the Internet at http://pubs.acs.org.
Figure 3. In vivo tagging of carrier protein fusion (VibB) within E. coli.
(a) Growth of E. coli culture (OD600) over a range in concentrations of 1.
∞ was measured at 1360 min. (b) In vivo formation of crypto-VibB
following a time course following addition of 1 mM 1 to culture. (c) In
vivo formation of crypto-VibB following a time course following addition
of 100 µM 1 to culture.
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While the percentage uptake of 1 is modest, the intracellular
concentration is sufficient to allow the labeled precursor to
incorporate into the CoA pathway. Following incubation of the co-
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