A fluorescent analogue of UDP-N-acetylglucosamine: Application for FRET assay of peptidoglycan translocase II (MurG)

Article abstract of DOI:10.1039/b313316h

A direct continuous fluorescence assay for translocase II MurG based on fluorescence resonance energy transfer (FRET) has been developed using a 6-substituted fluorescent analogue of UDP-N-acetylglucosamine.

Full text of DOI:10.1039/b313316h

A fluorescent analogue of UDP-N-acetylglucosamine: application for
FRET assay of peptidoglycan translocase II (MurG)
Jian-Jun Li and Timothy D. H. Bugg*
Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL.
E-mail: T.D.Bugg@warwick.ac.uk; Fax: 02476-524112; Tel: 02476-573018
Received (in Cambridge, UK) 22nd October 2003, Accepted 24th November 2003
First published as an Advance Article on the web 9th December 2003
A direct continuous fluorescence assay for translocase II MurG
based on fluorescence resonance energy transfer (FRET) has
been developed using a 6-substituted fluorescent analogue of
UDP-N-acetylglucosamine.
FRET involves the transfer of fluorescence energy from a donor
to an acceptor in the substrate or product of the reaction. FRET has
become an important technique for investigating a variety of
biological phenomena that produce changes in molecular prox-
imity.8 We have previously used a UDPMurNAc-pentapeptide
substrate modified with a dansyl fluorophore at position 3
(modified on N of meso-DAP) as a fluorescent substrate for the
preceding enzyme translocase I (MraY).9 We have therefore
investigated the possibility of attaching a fluorophore to the
UDPGlcNAc substrate of MurG, in order to set up a FRET assay for
MurG. We have used an indole-3-acetic acid fluorophore (lem 345
nm), which is able to transfer fluorescence energy effectively to the
dansyl fluorophore (lex 340 nm, lem 500 nm).
Functionalisation of UDP-GlcNAc was achieved by Pt/O2
oxidation of the C-6 hydroxyl of UDP-GlcNAc to a carboxylic acid,
using the method of Yamazaki et al.10 1-(3-(Dimethylamino)pro-
pyl)-3-ethylcarbodiimide (EDC) coupling of amines to the C-6
carboxylic acid was unsuccessful, but coupling of hydrazides at pH
4.75 proceeded smoothly, using the method of Pouyani et al.11
Indole-3-acetic hydrazide was successfully attached to oxidized
UDP-GlcNAc in 63% yield (Scheme 1). Using adipic hydrazide, a
further indole-acetic acid derivative containing a 10-atom linker
was also synthesized.
The glycosyltransferase enzyme translocase II (MurG) catalyses
the second step of the lipid cycle of bacterial peptidoglycan
biosynthesis, namely the transfer of a GlcNAc residue from UDP-
N-acetylglucosamine (UDP-GlcNAc) to undecaprenyl pyrophos-
phoryl MurNAc-pentapeptide (lipid intermediate I) to form un-
decaprenyl pyrophosphoryl GlcNAc-MurNAc-pentapeptide (lipid
intermediate II) (Fig. 1).1 Enzymes of this pathway are important
targets for antibiotic chemotherapy.1 MurG is known to be
inhibited by the cyclic peptide ramoplanin.2 The availability of an
X-ray crystal structure for MurG makes it an attractive target for
drug development,3 but assay of this enzyme is difficult, due to the
complexity of the substrates.
Since lipid I is not readily isolable from bacterial cells, and is
difficult to handle, some lipid I analogues have been synthesized.4–7
Generally, a radiochemical assay is the only method for measure-
ment of MurG activity.4,6 Recently a coupled assay has been
reported, in which the release of UDP was detected by coupling
with pyruvate kinase/lactate dehydrogenase.5,7 In this communica-
tion, we report a direct continuous fluorescence assay for MurG
based on fluorescence resonance energy transfer (FRET).
Escherichia coli MurG was purified from overexpressing
construct pET3a carrying the murG gene.4 UDPMurNAc-
L
-Ala-
D-
Glu-m-DAP (Ne-dansyl)-
D-Ala-
D-Ala was prepared as previously
described,9 and was converted to dansyl-labelled lipid I having C35
prenyl chain12 by treatment with E. coli translocase I, followed by
extraction into n-butanol.13 The fluorescent UDP-GlcNAc ana-
logues14 were tested as substrates for MurG, in the presence of
dansyl-labelled lipid I, in 0.2 M Tris-HCl buffer (pH 7.5)
containing 10 mM MgCl2, 0.2% CHAPS. Time-dependent fluores-
cence changes were observed (lex 290 nm, lem 500 nm), dependent
upon both substrate and enzyme, as shown in Fig. 2.
A linear relationship between enzyme concentration and rate of
increase in fluorescence was observed (Fig. 3). No fluorescence
changes were observed in the absence of enzyme. Turnover of lipid
I to lipid II was further confirmed by measurement of UDP release,
using a coupled assay,5,7 and by thin layer chromatography.
Interestingly, the two fluorescent analogues demonstrated different
behaviour: 1 showed 1.5-fold higher rates of fluorescence increase
than 2, implying a more efficient energy transfer in 1, due to the
shorter linker.
Using the dansyl-labelled lipid I and 1 as substrates, the kinetic
parameters for processing by MurG were determined using a
Fig. 1 MurG catalysed reaction. In the FRET assay, the dansyl fluorophore
(F1) acceptor is attached to position 3 of pentapeptide, the indole-3-acetic
acid fluorophore (F2) donor is attached to the C-6 position of UDP-
GlcNAc.
Scheme 1 Synthesis of fluorescent analogues of UDP-GlcNAc.
182
C h e m . C o m m u n . , 2 0 0 4 , 1 8 2 – 1 8 3
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

thank Dr S. Donadio (Vicuron Pharmaceuticals, Milan, Italy) for
the provision of a sample of ramoplanin.
Notes and references
1 T. D. H. Bugg, in Comprehensive Natural Products Chemistry, ed. M.
Pinto, Elsevier, Oxford, 1999, vol. 3, p. 241.
2 (a) E. A. Somner and P. E. Reynolds, Antimicrob. Agents Chemother.,
1990, 34, 413; (b) P. E. Reynolds and E. A. Somner, Drugs Exptl. Clin.
Res., 1990, 16, 385.
Fig. 2 Progress of MurG catalysed reaction upon addition of 3.0 mg MurG
to 3.0 mM dansyl lipid I and 2.7 mM 1.
3 S. Ha, D. Walker, Y. Shi and S. Walker, Protein Sci., 2000, 9, 1045.
4 S. Ha, E. Chang, M. C. Lo, H. B. Men, P. Park, M. Ge and S. Walker,
J. Am. Chem. Soc., 1999, 121, 8415.
5 L. Chen, H. B. Men, S. Ha, X. Y. Ye, L. Brunner, Y. N. Hu and S.
Walker, Biochemistry, 2002, 41, 6824.
6 G. Auger, J. van Heijenoort, D. Mengin-Lecreulx and D. Blanot, FEMS
Microbiol. Lett., 2003, 219, 115.
7 H. Liu, T. K. Ritter, R. Sadamoto, P. S. Sears, M. Wu and C.-H. Wong,
ChemBioChem, 2003, 4, 603.
8 R. P. Haughland, Handbook of Fluorescent Probes and Research
Chemicals, 6th Edn., Molecular Probes, Eugene, OR, USA, 1996, p.
46.
9 P. E. Brandish, M. K. Burnham, J. T. Lonsdale, R. Southgate, M. Inukai
and T. D. H. Bugg, J. Biol. Chem., 1996, 271, 7609.
10 T. Yamazaki, C. D. Warren, A. Herscovics and R. W. Jeanloz, Can. J.
Chem., 1981, 59, 2247.
Fig. 3 Relative rate of increase in fluorescence against enzyme concen-
tration.
Lineweaver–Burk plot. The measured Km value for 1 was 0.06 mM,
much lower than the reported Km value for unmodified UDP-
GlcNAc (58 mM4), in which case radiochemical assay was used.
The measured kcat value was 153 Fl units/mg protein/min.
Measurement of UDP release using the coupled assay gave a rate of
turnover of 0.025 mmoles/mg protein/min. Fluorescence changes
could be totally inhibited by 20 mM ramoplanin, and 40% inhibited
by 0.02 mM ramoplanin.
A number of other detergents were examined for activity in this
assay. Fluorescence changes were only observed using zwitterionic
detergents (CHAPS, SB3–10), at detergent concentrations below
the critical micellar concentration (CMC). Therefore it appears that
the reaction is not taking place in a detergent micelle, but that
probably the low concentration of detergent is required to solubilise
the lipid I substrate.
In conclusion, UDP-GlcNAc was successfully modified through
the C-6 position with a fluorescent label. Assays with E. coli MurG
have shown that the enzyme can accept the modified UDP-GlcNAc
as substrate, and energy transfer is observed under certain
conditions with a dansyl-labelled lipid I substrate. To our
knowledge, this is the first direct continuous fluorescence assay for
MurG.15 The availability of fluorescently modified UDP-GlcNAc
will enable fluorescent assays to be established for other glycosyl-
transferases that use UDP-GlcNAc.
We would like to thank GlaxoSmithKline Pharmaceuticals for
financial support, and Dr M. Burnham and Dr K. O’Dwyer for
helpful comments. We thank Professor S. Walker for the provision
of expression construct pET3a carrying the murG gene, and we
11 T. Pouyani and G. D. Prestwich, Bioconjugate Chem., 1994, 5, 339.
12 Auger et al. and Chen et al. found lipid I bearing a C55 prenyl chain is
a poor substrate for MurG. See refs. 5 and 6.
13 J. S. Anderson, M. Matsuhashi, M. A. Haskin and J. L. Strominger, J.
Biol. Chem., 1967, 242, 3180.
14 The fluorescent analogues of UDP-GlcNAc 1 and 2 were characterised
by 1H NMR and MS. 1, dH (400 MHz, D2O), 7.75 (d, 1H, J = 8.3 Hz,
H-6 uracil), 7.61 (d, 1H, J = 8 Hz, H-4 indole), 7.47 (d, 1H, J = 8.0 Hz,
H-7 indole), 7.32 (s, 1H, H-2 indole), 7.21 (dd, 1H, J = 7.3, 7.0 Hz, H-6
indole), 7.13 (dd, J = 7.1, 7.0 Hz, H-5 indole), 5.82 (d, 1H, J = 3.5 Hz,
H-1 ribose), 5.81 (d, 1H, J = 8.3 Hz, H-5 uracil), 5.51 (dd, 1H, JPH
=
7.4 Hz, JHH = 3.2 Hz, H-1 hexose), 4.38 (d, 1H, J = 10.0 Hz, H-5
hexose), 4.17 (m, 2H, H-2, 3 ribose), 4.11 (m, 2H, H-5a, 5b ribose), 4.06
(m, 1H, H-4 ribose), 4.01 (dt, 1H, J = 10.3, 3.0 Hz, H-2 hexose), 3.82
(s, 2H, CH2-indole), 3.81 (t, 1H, J = 9.8 Hz, H-3 hexose), 3.70 (t, 1H,
J = 9.4 Hz, H-4 hexose), 2.03 (s, 3H, NAc). ESI-MS (M 2 1) 791.16,
expected 792.14; 2, dH (400 MHz, D2O), 7.85 (d, 1H, J = 8.8 Hz, H-6
uracil), 7.59 (d, 1H, J = 7.8 Hz, H-4 indole), 7.45 (d, 1H, J = 8.0 Hz,
H-7 indole), 7.30 (s, 1H, H-2 indole), 7.20 (dd, 1H, J = 7.0, 7.0 Hz, H-6
indole), 7.12 (dd, 1H, J = 7.8, 7.0 Hz, H-5 indole), 5.87 (d, 1H, J = 4.2
Hz, H-1 ribose), 5.85 (d, 1H, J = 8.0 Hz, H-5 uracil), 5.51 (dd, 1H, JPH
= 7.3, JHH = 3.0 Hz, H-1 hexose), 4.36 (d, 1H, J = 9.8 Hz, H-5
hexose), 4.26 (m, 2H, H-2, 3 ribose), 4.20 (m, 2H, H-5a, 5b ribose), 4.12
(m, 1H, H-4 ribose), 4.00 (dt, 1H, J = 10.5, 3.0 Hz, H-2 hexose), 3.80
(t, 1H, J = 9.7 Hz, H-3 hexose), 3.79 (s, 2H, CH2-indole), 3.69 (t, 1H,
J = 9.5 Hz, H-4 hexose), 2.31–2.25 (m, 4H, CH2CH2CH2CH2), 2.01 (s,
3H, NAc), 1.62–1.57 (m, 4H, CH2CH2CH2CH2). ESI-MS (M 2 1)
933.25, expected 934.21.
15 During the preparation of this manuscript, a fluorescent binding assay
for MurG has been published. See: J. S. Ham, Y. Hu, L. Chen, B. Gross
and S. Walker, J. Am. Chem. Soc., 2003, 125, 11168.
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183