In vitro reconstitution of the radical S-adenosylmethionine enzyme MqnC involved in the biosynthesis of futalosine-derived menaquinone

Article abstract of DOI:10.1021/bi400498d

The radical S-adenosylmethionine enzyme MqnC catalyzes conversion of dehypoxanthine futalosine (DHFL) to the unique spiro compound cyclic DHFL in the futalosine pathway for menaquinone biosynthesis. This study describes the in vitro reconstitution of [4Fe-4S] cluster-dependent MqnC activity and identifies the site of abstraction of a hydrogen atom from DHFL by the adenosyl radical.

Full text of DOI:10.1021/bi400498d

Rapid Report
pubs.acs.org/biochemistry
In Vitro Reconstitution of the Radical S‑Adenosylmethionine Enzyme
MqnC Involved in the Biosynthesis of Futalosine-Derived
Menaquinone
Lisa E. Cooper,† Dmytro Fedoseyenko,† Sameh H. Abdelwahed,† Soong-Hyun Kim,† Tohru Dairi,‡
and Tadhg P. Begley*,†
†Department of Chemistry, Texas A&M University, College Station, Texas 77842, United States
‡Graduate School of Engineering, Hokkaido University, Sapporo, Japan
S
* Supporting Information
pathway for production of menaquinone (6). The early and late
ABSTRACT: The radical S-adenosylmethionine enzyme
MqnC catalyzes conversion of dehypoxanthine futalosine
(DHFL) to the unique spiro compound cyclic DHFL in
the futalosine pathway for menaquinone biosynthesis. This
study describes the in vitro reconstitution of [4Fe-4S]
cluster-dependent MqnC activity and identifies the site of
abstraction of a hydrogen atom from DHFL by the
adenosyl radical.
steps in the pathway are currently unknown and under
investigation. Humans and commensal intestinal bacteria lack
this pathway; therefore, it represents an attractive target for the
development of chemotherapeutic compounds.
Enzymes belonging to the radical SAM superfamily are
typically characterized by the presence of a cysteine-rich
CX3CX2C motif that ligates a [4Fe-4S] cluster.5,6 The iron−
sulfur cluster is responsible for the reductive cleavage of SAM,
generating a 5′-deoxyadenosyl (5′-dA) radical and methionine.
Amino acid sequence analysis revealed that MqnC contains this
characteristic CX3CX2C iron−sulfur cluster motif. Therefore, a
recombinant plasmid encoding mqnC from Bacillus halodurans
C-125 was introduced into E. coli BL21(DE3)-T1R containing
the E. coli suf operon for coexpression of His6-MqnC with
iron−sulfur cluster biogenesis proteins.7 Anaerobic purification
by nickel affinity chromatography yielded pure brown protein
with three irons and two sulfides per monomer, suggesting
incomplete reconstitution of the cluster. The UV−vis spectrum
of MqnC had an absorbance maximum at approximately 410
nm that disappeared upon exposure of the protein to oxygen
(Figure 1A).8 Electron paramagnetic resonance (EPR) spectra
and g values for MqnC treated with excess sodium dithionite
were consistent with a protein-bound [4Fe-4S]+ cluster (Figure
1B).
enaquinone (MK, vitamin K2) is a lipid-soluble molecule
M
that shuttles electrons between membrane-bound
protein complexes in the respiratory chain. In the classic
Escherichia coli pathway, it is biosynthesized from chorismate by
eight enzymes (MenA−MenH).1−3 Recently, an alternative
route for menaquinone production was discovered, termed the
futalosine pathway (Scheme 1).4 Chorismate (1)-derived
Scheme 1. Menaquinone Biosynthesis via the Futalosine
Pathway
Figure 1. Spectroscopic properties of MqnC. (A) UV−vis absorbance
of purified iron−sulfur cluster-reconstituted MqnC (160 μM) in 100
mM phosphate buffer (pH 7.5) before (blue) and after (red) exposure
to oxygen. (B) EPR spectra of purified iron−sulfur cluster-
reconstituted MqnC (280 μM) treated with (red and green) or
without (blue) dithionite.
futalosine (2) is converted to dehypoxanthine futalosine
(DHFL, 3) by futalosine hydrolase MqnB, and 3 is transformed
into cyclic dehypoxanthine futalosine (CDHFL, 4) by the
radical S-adenosylmethionine (SAM) enzyme MqnC. MqnD
then converts 4 to 1,4-dihydroxy-6-naphthoic acid (5). In the
later steps of the pathway, on the basis of the annotation and
clustering of the open reading frames in Streptomyces coelicolor
A3(2), it is possible that SCO4491 (prenylation) and SCO4556
(methylation) could be involved in completing the biosynthetic
Received: April 21, 2013
Revised: June 6, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/bi400498d | Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Rapid Report
On the basis of gene disruption studies in S. coelicolor, MqnC
is predicted to catalyze the conversion of 3 to 4.4 To test the
activity of MqnC, DHFL was synthesized as illustrated in
Scheme 2 (experimental methods are described in detail in the
These results confirm the predicted role of MqnC as the DHFL
cyclase.
Deuterium labeling of DHFL was used to elucidate the site of
hydrogen atom abstraction by the 5′-dA radical (13) during
MqnC catalysis. DHFL that was site-specifically deuterated at
C4′ was synthesized and used as a substrate for MqnC. The
transfer of deuterium from [4-2H]-DHFL to 13 was monitored
by LC−MS (Figure 3). The data revealed an increase of a single
Scheme 2. Synthesis of DHFL and [4-2H]-DHFL
Supporting Information). When MqnC was incubated under
anaerobic conditions with sodium dithionite, DHFL, and SAM
(11) in phosphate buffer (pH 7.5), the production of CDHFL
and 5′-deoxyadenosine (5′-dAd) was observed by high-
performance liquid chromatography (HPLC) as confirmed by
co-elution with authentic standards (Figure 2A). CDHFL was
Figure 3. MqnC-mediated hydrogen atom abstraction by the 5′-dA
radical occurs at C4′ of DHFL. (A) Mass spectrum demonstrating
66% deuterated [4-2H]-DHFL. (B) 5′-dAd derived from the MqnC
reaction using DHFL as the substrate. (C) 5′-dAd derived from the
MqnC reaction using [4-2H]-DHFL as the substrate. Calculated exact
masses ([M − H]−) of DHFL, [4-2H]-DHFL, and 5′-dAd: m/z 295.1,
296.1, and 250.1, respectively.
mass unit for 5′-dAd, thus demonstrating that the hydrogen
from C4′ of DHFL is abstracted by the 5′-dA radical. The low
efficiency of label transfer is most likely due to the substantial
levels of uncoupled 5′-dAd formation [10:1 (Figure S5 of the
Supporting Information)] as well as a primary isotope effect on
the initial hydrogen atom abstraction, which would favor
consumption of [4-1H]-DHFL over [4-2H]-DHFL.
A mechanistic proposal for the MqnC-catalyzed reaction is
outlined in Scheme 3. Reductive cleavage of SAM generates
adenosyl radical 13, which then abstracts a hydrogen atom from
DHFL 3 to give radical 15. Cyclization gives 16, which after
deprotonation transfers an electron back to the [4Fe-4S]2+
cluster to give CDHFL 4.
In summary, the activity of the radical SAM enzyme MqnC
was successfully reconstituted. EPR studies confirmed that
MqnC contains a [4Fe-4S] cluster, and isotope labeling studies
demonstrated that the C4′ hydrogen atom of DHFL 3 is
abstracted by the 5′-deoxyadenosyl radical. A mechanistic
proposal for the MqnC-catalyzed reaction is described.
Experiments are in progress to test this proposal.
Figure 2. MqnC activity in vitro. (A) HPLC chromatograms
monitoring absorbance at 250 nm. (B) Mass spectrum for CDHFL
purified from the S. coelicolor mqnD-disruptant strain. (C) Mass
spectrum for CDHFL from the full MqnC reaction. Calculated exact
mass of CDHFL of m/z 293.1 ([M − H]−).
not observed in control reaction mixtures from which sodium
dithionite, DHFL, SAM, or MqnC had been omitted. Assay and
control samples were also analyzed by liquid chromatography
and mass spectrometry (LC−MS) using an Agilent 1200 liquid
chromatograph (ChemStation) with a Bruker Daltonics
micrOTOF-Q II ESI-Qq-TOF mass spectrometer (HyStar).
The observed mass for cyclic DHFL in negative ion mode was
m/z 293.1 as compared to the theoretical exact mass of m/z
293.1 [[M − H]− (Figure 2C)]. The authentic standard for
CDHFL was purified from the previously described S. coelicolor
SCO4326-disruptant strain (observed mass of m/z 293.1).4
B
dx.doi.org/10.1021/bi400498d | Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Rapid Report
of the model actinomycete Streptomyces coelicolor A3(2). Nature 417,
141−147.
Scheme 3. Proposed MqnC Reaction Mechanism
(3) Dairi, T., Kuzuyama, T., Nishiyama, M., and Fujii, I. (2011)
Convergent strategies in biosynthesis. Nat. Prod. Rep. 28, 1054−1086.
(4) Hiratsuka, T., Furihata, K., Ishikawa, J., Yamashita, H., Itoh, N.,
Seto, H., and Dairi, T. (2008) An alternative menaquinone
biosynthetic pathway operating in microorganisms. Science 321,
1670−1673.
(5) Duin, E. C., Lafferty, M. E., Crouse, B. R., Allen, R. M., Sanyal, I.,
Flint, D. H., and Johnson, M. K. (1997) [2Fe-2S] to [4Fe-4S] cluster
conversion in Escherichia coli biotin synthase. Biochemistry 36, 11811−
11820.
(6) Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F., and
Miller, N. E. (2001) Radical SAM, a novel protein superfamily linking
unresolved steps in familiar biosynthetic pathways with radical
mechanisms: Functional characterization using new analysis and
information visualization methods. Nucleic Acids Res. 29, 1097−1106.
́
(7) Hanzelmann, P., Hernandez, H. L., Menzel, C., García-Serres, R.,
̈
Huynh, B. H., Johnson, M. K., Mendel, R. R., and Schindelin, H.
(2004) Characterization of MOCS1A, an oxygen-sensitive iron-sulfur
protein involved in human molybdenum cofactor biosynthesis. J. Biol.
Chem. 279, 34721−34732.
(8) Lee, K. H., Saleh, L., Anton, B. P., Madinger, C. L., Benner, J. S.,
Iwig, D. F., Roberts, R. J., Krebs, C., and Booker, S. J. (2009)
Characterization of RimO, a new member of the methylthiotransferase
subclass of the radical SAM superfamily. Biochemistry 48, 10162−
10174.
ASSOCIATED CONTENT
* Supporting Information
Detailed procedures for cloning the mqnC expression construct,
MqnC expression and purification, DHFL synthesis, CDHFL
isolation, and HPLC and LC−MS methods. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Department of Chemistry, Texas A&M University, College
Station, TX 77842. E-mail: begley@chem.tamu.edu. Phone:
(979) 862-4091.
■
Author Contributions
L.E.C. was responsible for the biochemical studies. D.F., S.H.A.,
and S.-H.K. synthesized DHFL. L.E.C. and T.P.B. designed the
experiments and prepared the manuscript.
Funding
This research was supported by National Institutes of Health
Grant DK44083 to T.P.B. and Robert A. Welch Foundation
Grant A-0034.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Cynthia L. Kinsland (Cornell University, Ithaca, NY)
for the construction of pTHT-mqnC, Professor Tohru Dairi
(Hokkaido University) for providing the mqnD-disruptant
strain, and Professor Paul A. Lindahl (Texas A&M University)
for use of the EPR spectrometer.
REFERENCES
■
(1) Meganathan, R. (2010) Menaquinone/Ubiquinone Biosynthesis
and Enzymology. In Comprehensive Natural Products II: Chemistry and
Biology (Mander, L., and Liu, H.-W., Eds.) pp 411−444, Elsevier Ltd.,
Amsterdam.
(2) Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M., Challis, G.
L., Thomson, N. R., James, K. D., Harris, D. E., Quail, M. A., Kieser,
H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C. W.,
Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., Hornsby, T.,
Howarth, S., Huang, C. H., Kieser, T., Larke, L., Murphy, L., Oliver, K.,
O’Neil, S., Rabbinowitsch, E., Rajandream, M. A., Rutherford, K.,
Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S.,
Taylor, K., Warren, T., Wietzorrek, A., Woodward, J., Barrell, B. G.,
Parkhill, J., and Hopwood, D. A. (2002) Complete genome sequence
C
dx.doi.org/10.1021/bi400498d | Biochemistry XXXX, XXX, XXX−XXX