Specific biotinylation of IMP dehydrogenase

Article abstract of DOI:10.1016/j.bmcl.2011.01.042

IMP dehydrogenase (IMPDH) catalyzes a critical step in guanine nucleotide biosynthesis. IMPDH also has biological roles that are distinct from its enzymatic function. We report a biotin-linked reagent that selectively labels IMPDH and is released by dithiothreitol. This reagent will be invaluable in elucidating the moonlighting functions of IMPDH.

Full text of DOI:10.1016/j.bmcl.2011.01.042

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1363–1365
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Specific biotinylation of IMP dehydrogenase
B. Christopher Hoefler a, , Deviprasad R. Gollapalli b, Lizbeth Hedstrom b,c,
⇑
a Graduate Program in Biochemistry, Brandeis University, MS 009, 415 South St., Waltham, MA 02454, USA
b Department of Biology, Brandeis University, MS 009, 415 South St., Waltham, MA 02454, USA
c Department of Chemistry, Brandeis University, MS 009, 415 South St., Waltham, MA 02454, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
IMP dehydrogenase (IMPDH) catalyzes a critical step in guanine nucleotide biosynthesis. IMPDH also has
biological roles that are distinct from its enzymatic function. We report a biotin-linked reagent that selec-
tively labels IMPDH and is released by dithiothreitol. This reagent will be invaluable in elucidating the
moonlighting functions of IMPDH.
Received 6 December 2010
Revised 7 January 2011
Accepted 11 January 2011
Available online 14 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
IMPDH
Enzyme inhibitors
Nucleotide biosynthesis
6-Cl-purine ribotide
Biotin probe
Mechanism-based probe
Inosine 50-monophosphate dehydrogenase (IMPDH) catalyzes
the penultimate step of guanine nucleotide biosynthesis, the
oxidation of IMP to XMP with the concomitant reduction of
NAD+1. IMPDH is up-regulated in rapidly dividing cells, and inhibi-
tion of IMPDH blocks proliferation and induces differentiation.
IMPDH-targeted drugs are used in anticancer, antiviral, and immu-
nosuppressive therapy, and are in development for antimicrobial
applications. Some recent observations suggest additional
functions for IMPDH beyond its well-established role in purine
nucleotide biosynthesis. IMPDH associates with telomeric se-
quences, polyribosomes and actively transcribing promoters, local-
izes to lipid vesicles in response to insulin and aggregates in
response to guanine nucleotide depletion.2–6 Clearly many ques-
tions remain regarding the biological function of this housekeeping
enzyme: is IMPDH enzymatically active in these different cellular
contexts? what factors interact with IMPDH in each location?
how do these interactions change in response to insulin and other
signals?
For example, 6-Cl-purine ribotide (6-Cl-PRT) reacts with the con-
served Cys to form a stable 6-linked adduct (Fig. 1).9,12,15 We
exploited this unusual reactivity to create a reagent that specifi-
cally biotinylates IMPDH by attaching a biotin to the 20 position
of 6-Cl-PRT via an aminopropyl linker to produce 6-Cl-PRT-biotin
(1, Fig. 1). The synthetic route started with xylofuranose and is de-
scribed in the Supplementary data. Importantly, the reagents are
relatively inexpensive, the products easy to purify and each step
is high-yielding, and scalable.
The reactions of 6-Cl-PRT and 6-Cl-PRT-biotin were character-
ized with IMPDHs from various organisms already in hand
(Fig. 2).16 6-Cl-PRT rapidly inactivates E. coli IMPDH, and the
presence of the biotin has no significant effect on this reaction
(kinact/Ki = 120 Mꢀ1 sꢀ1 and 130 Mꢀ1 sꢀ1 for 6-Cl-PRT and 6-Cl-
PRT-biotin, respectively, at 25 °C; standard errors are within 20%
A mechanism-based probe would be an ideal tool to address
these questions.7,8 The IMPDH reaction is unusual among the
enzymes involved in nucleotide metabolism in that it involves
the nucleophillic attack of a conserved Cys residue, and several
nucleotide analogs inactivate IMPDH by modifying this Cys.1,9–14
⇑
Corresponding author. Tel.: +1 781 736 2333, fax: +1 781 736 2349.
E-mail address: hedstrom@brandeis.edu (L. Hedstrom).
Texas A&M University, College of Agriculture & Life Sciences, Department of
Biochemistry and Biophysics, 103 Biochemistry Bldg., 2128 TAMU, College Station,
TX 77843-2128, USA.
Figure 1. Reaction of 6-Cl-PRT and 6-Cl-PRT-biotin with IMPDH.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.042

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B. C. Hoefler et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1363–1365
Figure 2. Inactivation of human IMPDH type 2 by 6-Cl-PRT-biotin at 37 °C. A. Loss
of IMPDH activity over time incubated with 6-Cl-Purine-biotin, (s) 100 M 6-Cl-
PRT-biotin, (+) 200 M 6-Cl-PRT-biotin. B. Rate of
l
l
M 6-Cl-PRT-biotin, (}) 500 l
inactivation of IMPDH as a function of 6-Cl-PRT-biotin concentration.
Figure 4. Selectivity of 6-Cl-PRT-biotin. Lysates of wild-type E. coli strain MG1655
and guaB, which lacks endogenous IMPDH, were treated with 6-Cl-PRT-biotin and
D
analyzed as described in Figure 3.
for all values unless otherwise noted). 6-Cl-PRT is a less effective
inactivator of human IMPDH type 2 (hIMPDH2), with kinact/Ki =
0.9 Mꢀ1 sꢀ1 at 25 °C; hIMPDH2 has a lower specific activity than
E. coli IMPDH,1 so this reduced activity is not surprising. The addi-
tion of the biotin reduces kinact/Ki by at least a factor of 10. Inacti-
vation of hIMPDH2 by 6-Cl-PRT-biotin is readily observed at 37 °C
(kinact/Ki = 1.4 Mꢀ1 sꢀ1), so this reagent will be useful for probing
IMPDH in human cells.
To determine if 6-Cl-PRT-biotin covalently labels IMPDH as
designed, the inactivated enzymes were analyzed by denaturing
SDS–PAGE, transferred to PVDF membranes and probed with avi-
din-horse radish peroxidase, which binds specifically to biotin.17
Strong labeling of IMPDH was observed, demonstrating that a
covalent adduct is formed (Fig. 3). No labeling occurred when
IMPDH was denatured prior to treatment with 6-Cl-PRT-biotin
(not shown), and labeling was reduced by a factor of >2000 when
the active site Cys was replaced with Ala (Fig. 3A). These observa-
tions demonstrate that 6-Cl-PRT-biotin selectively modifies the
active site Cys of IMPDH as designed.
In the course of these experiments, we noticed that labeling was
less efficient in the presence of dithiothreitol. Preincubation of
6-Cl-PRT with dithiothreitol had no effect on the labeling reaction,
demonstrating that the probe is stable under these conditions. In
contrast, treatment of 6-Cl-PRT-modified IMPDH with dithiothrei-
tol released the label (Fig. 3B). These experiments establish mild
conditions to break the 6-S-enzyme-PRT adduct which will be very
useful in isolating IMPDH complexes.
lower molecular weight in the
DguaB strain. 6-Cl-PRT is also
known to inactivate GMP reductase (GMPR),6 an enzyme closely
related to IMPDH with a similar mechanism and nucleophillic
Cys. 6-Cl-PRT-biotin also labels GMPR (Fig. 4), so it is likely that
the smaller band is GMPR. IMPDH and GMPR have different molec-
ular weights and are induced under very different conditions, so
this cross-reactivity should not limit the usefulness of the probe.
Biotinylation is a widely used method for labeling biological
macromolecules for isolation and visualization.7,8 Here we have
described a reagent, 6-Cl-PRT-biotin, that selectively labels IMPDH
and can be removed under mild conditions. This reagent will be
invaluable in determining if the active site of IMPDH is accessible
when the protein is associated with polyribosomes, chromatin
and lipid vesicles as well as for pulldown experiments to identify
interacting factors. 6-Cl-PRT-biotin and related compounds may
also be useful in cellular visualization experiments, although it is
likely that a prodrug approach will be required to enable probe up-
take. We believe that 6-Cl-PRT-biotin will be an invaluable tool for
investigating the biological functions of this ‘enzyme of
consequence’ in virtually all organisms.
Acknowledgements
We thank Prof. Barry Snider for advice on the synthesis of
6-Cl-PRT-biotin. Funding was provided by NIH GM54403 and R21
EY017325.
To test the specificity of labeling, an E. coli lysate was treated
with 6-Cl-PRT-biotin and analyzed as described above (Fig. 4).
One major band is observed; this band is absent in a
DguaB strain
that lacks endogenous IMPDH, and can therefore be assigned to
IMPDH with confidence. A second very weak band is observed at
Supplementary data
Supplementary data (synthesis of 6-Cl-PRT and detailed exper-
imental procedures) associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.042.
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Figure 3. Labeling of IMPDH. A. Recombinant E. coli IMPDH was inactivated with
6-Cl-PRT-biotin and subjected to non-reducing SDS–PAGE, transferred to a PVDF
membrane, and probed with avidin-HRP. B. Purified recombinant human IMPDH
type 2 was treated with 200 lM 6-Cl-PRT-biotin. Half of the sample was then
treated with 6 mM DTT. Samples were desalted over Sephadex G-50, denatured
under non-reducing conditions, and separated by SDS–PAGE. After transfer to a
PVDF membrane, the blot was probed with avidin-HRP.

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of the inhibitor. The reaction was quenched by the addition of 2 mM IMP and
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16. Conditions: IMPDH was incubated in 50 mM Tris–HCl, pH 8, containing
100 mM KCl and 0.5 mM TCEP (assay buffer) with increasing concentrations
17. Denaturing SDS–PAGE was performed using standard methods except that
TCEP was substituted for dithiothreitol to preserve the thiol linkage. Samples
were denatured by heating at 100 °C for 3 min or by adding urea buffer.