A natural carbohydrate substrate for Mycobacterium tuberculosis methionine sulfoxide reductase A

Article abstract of DOI:10.1039/b817483k

Enzymatic reduction of the methylsulfinylxylofuranosyl (MSX) groups in lipoarabinomannan provides proof of the absolute configuration of MSX and a possible biochemical mechanism for oxidative protection in Mycobacterium tuberculosis. The Royal Society of

Full text of DOI:10.1039/b817483k

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A natural carbohydrate substrate for Mycobacterium tuberculosis
methionine sulfoxide reductase Aw
Susanne A. Stalford,a Martin A. Fascione,a Smitha J. Sasindran,b Delphi Chatterjee,c
Subramanian Dhandayuthapanib and W. Bruce Turnbull*a
Received (in Cambridge, UK) 6th October 2008, Accepted 30th October 2008
First published as an Advance Article on the web 11th November 2008
DOI: 10.1039/b817483k
Enzymatic reduction of the methylsulfinylxylofuranosyl (MSX)
groups in lipoarabinomannan provides proof of the absolute
configuration of MSX and a possible biochemical mechanism
for oxidative protection in Mycobacterium tuberculosis.
methionine residues in proteins could provide anti-oxidative
protection by sequestering peroxides through a redox cycle
(Scheme 1a) comprising oxidation by H2O2 and subsequent
enzymatic reduction by methionine sulfoxide reductase (Msr)
enzymes that are found in all organisms.13,14 Indeed, over-
expression of the methionine sulfoxide reductase A enzyme
(MsrA) in human fibroblasts can reduce the level of protein
carbonyl formation when challenged with H2O2.15 The MsrA
enzyme from Mycobacterium smegmatis has also been shown
to be important for survival of mycobacteria inside macro-
phage cells.16 MsrA is known to accommodate alternative
substrates bearing a methylthio group.17 Therefore, the struc-
tural similarities between methionine and MTX led us to
consider if a similar redox cycle could be possible in which
methionine is replaced by MTX (Scheme 1b). We also use this
oxidation/reduction mechanism to provide a stereochemical
proof for the absolute configuration of MTX.
Tuberculosis (TB) remains a major threat to world health,1
with 9.2 million new cases and 1.7 million deaths from TB in
2006.2 The disease is caused by Mycobacterium tuberculosis
(Mtb) which invades and colonises macrophage cells. The
ability of Mtb to survive within this inhospitable environment
has been attributed to its robust cell wall,3 that comprises
complex glycolipids including mycolyl-arabinogalactan-
peptidoglycan (mAGP)4 and lipoarabinomannan (LAM).5
LAM facilitates the entry of the bacterium into macrophages,5
prevents macrophage activation, and protects Mtb from
damage by superoxide and hydroxyl radicals.6 Although
macrophages also produce H2O2 during a respiratory burst,7
the effect of H2O2 on LAM was not reported alongside the
studies on superoxide and hydroxyl radicals.6
LAM was purified from the Mtb clinical isolate CSU20 as
described previously.8 1H NMR spectroscopy of CSU20-LAM
(Fig. 1a) indicated that there was approximately one MTX or
MSX residue for every 45 monosaccharide units. This ratio
corresponds to one xylofuranosyl residue per LAM molecule,
or one MTX/MSX for every 5–6 mannose caps.5,8 When one
considers the relative concentrations of proteins and LAM in
mycobacterial cell walls,18 MTX could be as prevalent as
methionine residues in this part of the cell. The presence of
two overlapping doublets (corresponding to H-1) at ca.
5.45 ppm indicated that the sample was already partially
oxidised to a 1 : 1 mixture of MSX diastereoisomers. The
occurrence of MSX in previous studies has been attributed to
oxidation during purification of the LAM.8 Upon addition of
excess H2O2 to the sample, the MTX anomeric signal
decreased in size to be replaced by the overlapping MSX
Recent structural studies have revealed two novel sugar
substituents on the terminal mannose caps of LAM,8,9 which
were identified as an a-5-methylthioxylofuranosyl (MTX)
group and its corresponding sulfoxide derivative (MSX)
(Scheme 1b).10,11 The cell surface location of MTX8 will make
it particularly exposed to damage by reactive oxygen species
(ROS) produced by macrophages. Oxidation of the sulfide
group would almost certainly modulate any functional inter-
actions between the sugar and other biomolecules. For exam-
ple, an MTX-a(1,4)-mannosyl disaccharide has been shown to
inhibit the production of cytokine TNF-a in the human
monocyte cell line THP-1,11 but the effect is reduced upon
oxidation of the sugar.
Conversely, sacrificial oxidation of MTX would sequester
ROS from solution and thus prevent other sensitive mole-
cules from oxidative damage. Levine has proposed12 that
a School of Chemistry and Astbury Centre for Structural Molecular
Biology, University of Leeds, Leeds, UK LS2 9JT.
E-mail: w.b.turnbull@leeds.ac.uk; Fax: +44 (0)113 343 6565;
Tel: +44 (0)113 343 7438
b University of Texas Health Science Center at San Antonio,
Department of Microbiology and Immunology, and Regional
Academic Health Center, 1214 West Schunior Street, Edinburg,
TX 78541, USA
c Department of Microbiology, Immunology, and Pathology, Colorado
State University, Fort Collins, CO 80523-1682, USA
w Electronic supplementary information (ESI) available: Experimental
details for oxidation and reduction experiments and for the prepara-
tion of methyl a-D- and -L-MSX. See DOI: 10.1039/b817483k
Scheme 1 Oxidation and reduction of (a) methionine and (b) MTX.
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Fig. 1 Anomeric (H-1) region of the 1H NMR spectra of LAM (a) before oxidation, and after successive treatment with (b) H2O2 and
(c) MsrA–DTT.
1
anomeric signals (Fig. 1b). It should be noted that no other
anomeric signals in the spectrum changed when the sample was
treated with H2O2. Therefore, it can be concluded that MTX is
oxidised selectively to MSX by H2O2, and no other mono-
saccharide component of LAM is capable of sequestering
H2O2. This observation was corroborated by experiments on
methyl a-mannopyranoside and methyl a/b-arabinofuranoside
which showed no oxidation by H2O2 under conditions
that allowed complete conversion of MTX to MSX (data not
shown).
catalytically active form of the enzyme. The H NMR spec-
trum of the reaction mixture showed that the low field MSX
anomeric signal disappeared to be replaced by the anomeric
signal for MTX (Fig. 1c). The MTX anomeric signal is slightly
larger than that for the remaining MSX as a consequence of
incomplete oxidation of the original MTX residues (Fig. 1b).
These results demonstrate that MSX is reduced stereo-
selectively by MsrA. A molecular model of methionine
sulfoxide docked into the crystal structure of Mtb MsrA
indicates that only the S-configured sulfoxide can be
accommodated in the enzyme active site.20 Therefore, we
would assign the remaining MSX signal to correspond to the
R-sulfoxide. Treatment of MSX-LAM with DTT alone had no
effect on the NMR spectrum, and hence did not modify the
polysaccharide structure.
Mycobacterium tuberculosis has two genes encoding Msr
enzymes: MsrA and MsrB which reduce S- and R-configured
sulfoxides, respectively. Cell wall/membrane and soluble
protein fractions were prepared according to Mueller-Ortiz
et al.19 Immunoblotting experiments showed that while MsrB
is confined to the cytosol of the bacterium, MsrA is present in
both the soluble and membrane/cell wall fractions (Fig. 2). As
deletion of the gene encoding MsrB is generally less important
for anti-oxidative protection than MsrA,14 we chose to focus
first on reduction of MSX by MsrA.
The stereoselective reduction of MSX presented us with an
opportunity to determine the absolute stereochemistry of
MTX while still attached to the polysaccharide. Inspection
of a model of MSX docked into the MsrA crystal structure
indicated that little of the MSX structure would be bound
within the enzyme active site (Supporting information
Fig. S1w). Therefore, either D- or L-configured MSX should
be able to act as a substrate for the enzyme, but in each case
only the S-configured sulfoxide should be reduced. The
remaining R-sulfoxides would be a pair of diastereoisomers
and would thus have different NMR spectra. We anticipated
that only one of these isomers would match the spectrum of
the natural polysaccharide.
MSX-LAM was treated with MsrA in the presence of excess
dithiothreitol (DTT) as a co-reductant to regenerate the
The methyl a-furanosides of D- and L-MTX were prepared
by chemical synthesis (see supporting informationw) and
oxidised to MSX by exposure to H2O2. No evidence for
over-oxidation to the sulfone was observed. The 1H NMR
Fig. 2 Immunoblot analysis of MsrA and MsrB in different fractions
of Mtb cells: (a) whole Mtb cell, (b) the cytosolic fraction and (c) the
cell wall/membrane fraction.
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and enzymatic reduction could also provide a mechanism for
more general anti-oxidative protection, as has been established
previously for methionine oxidation.12,15 Indeed, as H2O2 is a
direct precursor of hydroxyl radicals in vivo, sequestration of
H2O2 by MTX–MsrA could also reduce the production of
OHꢁ in the mycobacterial cell wall. Future studies will focus
on testing this hypothesis in vivo.
We acknowledge the Royal Society, the San Antonio Area
Foundation, the University of Leeds and NIH for funding.
D.C. is supported by NIH/NIAID AI 37139. W.B.T. is a
recipient of a Royal Society University Research Fellowship.
Mr J. Willis is thanked for assistance in protein production.
Notes and references
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6 J. Chan, X. Fan, S. W. Hunter, P. J. Brennan and B. R. Bloom,
Infect. Immun., 1991, 59, 1755.
Fig. 3 Anomeric (H-1) region of the 1H NMR spectra of methyl
a-D-MSX (a) before and (b) after treatment with MsrA–DTT.
Anomeric (H-1) region of the 1H NMR spectra of methyl a-L-MSX
(d) before and (c) after treatment with MsrA–DTT.
7 J. A. Imlay, Annu. Rev. Biochem., 2008, 77, 755.
8 A. Treumann, X. Feng, L. McDonnell, P. J. Derrick, A. E.
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Soc., 2006, 128, 5059.
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Natl. Acad. Sci. U. S. A., 1996, 93, 15036.
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spectra for D- and L-MSX were identical (Fig. 3a,d), as
expected for pairs of enantiomers. The difference in chemical
shift for the anomeric proton of methyl a-MSX from that of
MSX-LAM (Fig. 1b) can be attributed to the different anomeric
substituents.10 Upon treatment with MsrA and DTT, only the
S-sulfoxide isomers were reduced to give mixtures of MTX
and either the R-methylsulfinyl-D-xylofuranoside (Fig. 3b) or
R-methylsulfinyl-L-xylofuranoside (Fig. 3c).21 As the downfield
MSX anomeric signal is lost for both MSX-LAM (Fig. 1c) and
methyl a-D-MSX (Fig. 3b), we can thus conclude that MTX is
D-configured. This result provides independent corroboration
of the findings of Lowary and co-workers who compared the
NMR spectra of six synthetic MTX-mannosyl disaccharides
with the published NMR spectra of LAM.11
15 C. R. Picot, I. Petropoulos, M. Perichon, M. Moreau, C.
Nizard and B. Friguet, Free Radical Biol. Med., 2005, 39,
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17 J. Moskovitz, H. Weissbach and N. Brot, Proc. Natl. Acad. Sci.
U. S. A., 1996, 93, 2095.
In conclusion, we have demonstrated that exposure of LAM
to the biological oxidant H2O2 results in oxidation of only the
MTX substituent. Stereoselective reduction of the S-sulfoxide
isomer of MSX-LAM using MsrA provides proof of the
absolute configuration of this novel sugar without the need
to remove the sugar from the polysaccharide. While oxidation
of MTX would almost certainly affect its biological function
in vivo, this damage could be repaired, in part, by MsrA which
is present in the mycobacterial cell wall/membrane fraction.
Alternatively, there exists the possibility that MSX on the
surface of M. tuberculosis may be reduced by host MsrA and
MsrB when the bacteria reside inside macrophages. If so, then
MSX would be the first natural non-protein substrate for these
enzymes. Furthermore, this redox cycle of chemical oxidation
18 B. Hamasur, G. Kallenius and S. B. Svenson, FEMS Immunol.
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Med. Microbiol., 1999, 24, 11; I. Verma, A. Rohilla and
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20 A. B. Taylor, D. M. Benglis, Jr, S. Dhandayuthapani and
P. J. Hart, J. Bacteriol., 2003, 185, 4119.
21 X-ray crystallography could not be used to confirm the relative
configurations of the products as the sulfoxides were syrups.
Therefore, the configurations of the sulfoxides are assigned on
the basis of the known substrate specificity of MsrA (see ref. 13).
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112 | Chem. Commun., 2009, 110–112