Synthesis and application of isotopically labeled flavin nucleotides

Article abstract of DOI:10.1002/jlcr.3313

Flavin nucleotides, i.e. flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are utilized as prosthetic groups and/or substrates by a myriad of proteins, ranging from metabolic enzymes to light receptors. Isotopically labeled flavins have served as invaluable tools in probing the structure and function of these flavoproteins. Here we present an enzymatic synthesis of several radio- and stable-isotope labeled flavin nucleotides from commercially available labeled riboflavin and ATP. The synthetic procedure employs a bifunctional enzyme, Corynebacterium ammoniagenes FAD synthetase, that sequentially converts riboflavin to FMN and then to FAD. The final flavin product (FMN or FAD) is controlled by the concentration of ATP in the reaction. Utility of the synthesized labeled FAD cofactors is demonstrated in flavin-dependent thymidylate synthase. The described synthetic approach can be easily applied to the production of flavin nucleotide analogues from riboflavin precursors. Flavin nucleotides are utilized as prosthetic groups and/or substrates by a myriad of proteins. Here we present an enzymatic synthesis of several radio- and stable-isotope labeled flavin nucleotides. Utility of the synthesized labeled FAD cofactors is demonstrated in flavin-dependent thymidylate synthase. The described synthetic approach can be easily applied to the production of flavin nucleotide analogues from riboflavin precursors.

Full text of DOI:10.1002/jlcr.3313

Research article
Received 1 September 2014,
Revised 2 April 2015,
Accepted 6 June 2015
Published online 7 July 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3313
Synthesis and application of isotopically
labeled flavin nucleotides
a
Tatiana V. Mishaninaa,b and Amnon Kohen
*
Flavin nucleotides, i.e. flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are utilized as prosthetic groups
and/or substrates by a myriad of proteins, ranging from metabolic enzymes to light receptors. Isotopically labeled flavins
have served as invaluable tools in probing the structure and function of these flavoproteins. Here we present an enzymatic
synthesis of several radio- and stable-isotope labeled flavin nucleotides from commercially available labeled riboflavin and
ATP. The synthetic procedure employs a bifunctional enzyme, Corynebacterium ammoniagenes FAD synthetase, that
sequentially converts riboflavin to FMN and then to FAD. The final flavin product (FMN or FAD) is controlled by the
concentration of ATP in the reaction. Utility of the synthesized labeled FAD cofactors is demonstrated in flavin-dependent
thymidylate synthase. The described synthetic approach can be easily applied to the production of flavin nucleotide
analogues from riboflavin precursors.
Keywords: flavin adenine dinucleotide; FAD synthetase; isotopic labeling; thymidylate synthase
could be challenging to remove.17 In fact, commercially available
FMN synthesized this way contains ~30% of phosphorylated
impurities. Enzymatic phosphorylation by riboflavin kinase (also
Introduction
Flavins are incredibly versatile compounds capable of carrying out
one- and two-electron redox, nucleophilic and electrophilic
known as flavokinase, FMN synthetase, or ATP:riboflavin
chemistries.1,2 This versatility makes flavins invaluable as electron-
5′-phosphotransferase), on the other hand, yields pure FMN. A
carriers, prosthetic groups of proteins and even precursors in the
second enzyme, FAD synthetase (also known as FAD
biosynthesis of other biologically important molecules.3 The most
widely utilized flavins in nature are the ‘nucleotide’ forms of the
pyrophosphorylase, FMN adenylyl transferase or ATP:FMN
adenylyltransferase), can subsequently be used to adenylylate
vitamin riboflavin: flavin mononucleotide (FMN) and flavin adenine
FMN into FAD. This enzymatic FMN → FAD conversion (typical
yields 85–93%,18,19 based on the flavin) is much more efficient
dinucleotide (FAD), shown in Figure 1. Here we present the synthesis
of several flavin nucleotides isotopically labeled at the isoalloxazine
core or the adenyl tail, and their utility in analyzing an intermediate
of an enzymatic reaction. Isotopically labeled flavins and flavin
than the chemical routes to FAD (reported yields ranging from
6%20,21 to 14%,22 based on the flavin); however, it does require
preparation of an additional protein. The advantage of the
analogues have been used in the past to probe the structural
synthetic approach described here lies in using a single enzyme,
dynamics of light receptors,4,5 elucidate flavin transport and
Corynebacterium ammoniagenes FAD synthetase, to selectively
metabolism in healthy and diseased cells6,7 and gain insight into
convert riboflavin to either FMN or FAD, by varying the reagent
concentrations.
C. ammoniagenes FAD synthetase is a bifunctional enzyme that
the mechanisms of flavoproteins,8–10 among other applications. In
particular, reconstitution of cytochrome P450 reductase, the redox
partner of P450 enzymes, with FMN analogues shed light on the
combines the activities of
a
riboflavin kinase and
mechanism of electron transfer in this protein.11 FAD isotopologues
labeled with 13C in various positions of the isoalloxazine were
employed in ultrafast time-resolved infrared spectroscopy studies
of a Blue Light Using Flavin (BLUF)-domain protein AppA, a
photoreceptor.4,12 These studies demonstrated the direct response
of AppA protein matrix to the photoexcitation of flavin
chromophore, which affords a signaling state of the protein. Isotopic
labeling of the adenyl tail of FAD, on the other hand, may prove
useful in the flavoprotein systems where adenine is involved in
protein function, e.g. DNA photolyase in which FAD assumes an
unusual bent conformation and adenine mediates the electron
adenylyltransferase (Figure 2A). The recombinant enzyme has been
overexpressed in Escherichia coli and purified in the past, and its
steady-state kinetics have been characterized.23 Here we use
partially purified C. ammoniagenes FAD synthetase to produce
FAD isotopically labeled at either the adenyl tail or the isoalloxazine
core (Figure 2B) starting from commercially available isotopically
aDepartment of Chemistry, University of Iowa, Iowa City, IA, 52242-1727, United
States
transfer from isoalloxazine to the thymine dimer on DNA bDepartment of Biochemistry, University of Wisconsin-Madison, Madison, WI,
substrate.13,14
53706, United States
FMN is traditionally generated from riboflavin via chemical
*Correspondence to: Amnon Kohen, Department of Chemistry, University of
Iowa, E274 Chemistry Building, Iowa City, IA, United States.
phosphorylation of 5′ hydroxyl.15,16 However, this route invariably
generates isomeric monophosphates and bisphosphates, which E-mail: amnon-kohen@uiowa.edu
J. Label Compd. Radiopharm 2015, 58 370–375
Copyright © 2015 John Wiley & Sons, Ltd.

T. V. Mishanina and A. Kohen
(hydroxymethyl)aminomethane [Tris] base from Research Products
International Corp.
Expression and partial purification of FAD synthetase
C. ammoniagenes FAD synthetase was produced by E. coli following a
modified procedure of ref. 23. Bacteria were grown overnight at 30 °C
in 6-L Luria Broth medium containing 200 mg/L ampicillin. The cells
(34 g paste) were harvested and lysed by passing the cell suspension
through French press at 4 °C in Lysis Buffer [100 mL of 100 mM Tris,
pH 7.45, 10 mM EDTA, 1 mM DTT, 400 mM NaCl, 20 mM MgCl2,
3 mg/mL lysozyme, 0.1 mg/mL DNAase I, EDTA-free protease inhibitor
pellets (Roche)]. The cell debris was removed by centrifugation at
40 000 ×g, and the soluble fraction was treated with solid ammonium
sulfate to 50% saturation, i.e. 30.11 g solid ammonium sulfate added
per 100 mL solution at 4 °C. The precipitated proteins were pelleted
by centrifugation, and ammonium sulfate was added to the supernatant
to 80% saturation, i.e. an additional 19.98 g solid ammonium sulfate per
100 mL solution at 4 °C. After centrifugation, the pellet containing
partially purified FAD synthetase was dissolved in 36 mL of 100 mM Tris,
pH 7.45, 10 mM EDTA and 1 mM DTT, and then dialyzed against 1 L of
water for 1 h and finally against 1 L of 50 mM Tris, pH 7.45 overnight
at 4 °C. This crude preparation (total protein concentration of ca.
2.7 mg/mL as determined by Bradford assay)32 was used in the
synthesis of flavins.
Figure 1. Structure and atomic numbering of flavin adenine dinucleotide (FAD)
and shorter natural flavins.
labeled ATP or riboflavin, respectively. FMN labeled at isoalloxazine
can also be obtained via this route, by using stoichiometric
amounts of riboflavin and ATP in the synthetic mixture.
Following the description of the synthesis of FADs with four
different labeling patterns (Figure 2B), one application is
presented for the use of these labeled flavins in the mechanistic
studies of an enzyme, flavin-dependent thymidylate synthase
(FDTS). FDTS employs an FAD prosthetic group to reductively
methylate 2′-deoxyuridine-5′-monophosphate (dUMP) to 2′-
deoxythymidine-5′-monophosphate (dTMP), a DNA precursor,
in many human pathogens24–27 (Figure 3). FDTS presents an
Synthesis of labeled flavins
Reactions for the synthesis of labeled FAD contained the following:
50 μM riboflavin, 8 mM MgCl2, 1 mM ATP, 196 mM phosphocreatine,
800 units/mL of creatine phosphokinase in 50 mM Tris buffer, pH 7.6.
The reactions were initiated by addition of 100 μL partially purified
FAD synthetase (total protein concentration of ca. 2.7 mg/mL) per mL
exciting new target for antibiotics with low toxicity, considering of synthetic mixture and incubated at 25 °C until completion (typically
24 h), as determined by analytical HPLC. Radiolabeled FAD synthesis
was carried out in a final volume of 500 μL. For the synthesis of
compound 1, 50 μCi of tritiated ATP was used along with non-
radioactive ATP (final ATP concentration 1 mM, i.e. 20-fold excess over
riboflavin). Addition of unlabeled ATP was necessary to push the
reaction toward FAD production. As a result, the radioactive FAD yield
was quite low (~3%). However, unreacted 3H-ATP could be recovered
in the HPLC purification, lyophilized and re-used in additional cycles
of synthesis. In the synthesis of compound 3, 30 μCi of tritiated
riboflavin was employed in addition to non-radiolabeled riboflavin (final
that its mechanism of action differs drastically from ‘classical’
thymidylate synthase encoded by thyA gene in most organisms,
including humans.28,29 The details of FDTS chemical mechanism
are still under investigation, and our recent acid-trapping of an
intermediate in FDTS-catalyzed reaction has provided some
insight into the timing of chemical events in this enzyme.30
Surprisingly, a different derivative of an intermediate(s) is
trapped under basic conditions, which contains the pyrimidine
substrate plus an unknown adduct (Mishanina and Kohen,
unpublished data). The currently reported isotopically labeled total concentration 50 μΜ riboflavin). 13C,15N-labeled FAD was
synthesized in a final reaction volume of 10 mL with 13C,15N-labeled
FAD molecules are employed in efforts to identify this
mysterious adduct and add another piece to the mechanistic
puzzle of FDTS.
ATP (compound 2; no unlabeled ATP added to the synthesis mixture),
or 13C,15N-labeled riboflavin (compound 4; no unlabeled riboflavin
added to the synthesis mixture).
Analytical methods
Experimental
Separations were carried out on an Agilent 1100 series HPLC, with
UV/vis diode array detector, flow-scintillation analyzer (FSA, model
Materials
Chemicals were reagent grade and used as purchased unless specified RT505 from Packard, now PerkinElmer) for radioactive synthetic
otherwise. Unlabeled and [dioxopyrimidine-13C4,15N2]riboflavin, unlabeled
mixtures, or liquid scintillation counting (LSC, Tri-Carb model 2900 TR
and [13C4,15N2]ATP, phosphocreatine and creatine phosphokinase from
from PerkinElmer) for base-quenched FDTS reactions containing 3H-
rabbit muscle were obtained from Sigma-Aldrich. [2-14C]dUMP labeled FAD. An Ultima FloTM AP scintillation cocktail (PerkinElmer)
(53.2 mCi/mmol), [2,8-3H]ATP (32 Ci/mmol) and [7a,8a-3H]riboflavin was employed in flow-scintillation analyses and Ultima GoldTM in LSC.
(6.2 Ci/mmol) were purchased from Moravek Biochemicals. The FDTS from
An analytical reverse phase Supelco column (Discovery series
Thermatoga maritima (TM0449, GenBank accession number NP228259) 250 mm × 4.6 mm, 5 μm) was used at 0.8 mL/min for monitoring the
was expressed and purified as previously described.31 The E. coli progress of FAD synthetase reactions. The column was pre-equilibrated
expression system for C. ammoniagenes FAD synthetase was a gracious in 85:15 buffer:methanol mixture, and the following method was
gift from Prof. Dale E. Edmonson (Emory University). Magnesium chloride employed for separation of flavins (5 mM ammonium acetate buffer,
was purchased from BDH Chemicals, ammonium acetate, ammonium pH 6.5 as solvent A; methanol as solvent B): 0–5 min 15% B; 5–25 min
sulfate and sodium chloride from Fisher Scientific, and tris
15–75% B; 25–26 min 100% B. Elution of the flavins was followed by
J. Label Compd. Radiopharm 2015, 58 370–375
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

T. V. Mishanina and A. Kohen
Figure 2. (A) C. ammoniagenes FAD synthetase-catalyzed synthesis of flavin nucleotides from riboflavin, employed in current work. (B) Structures of the isotopically
labeled FAD molecules synthesized in this study.
Figure 3. Reaction catalyzed by flavin-dependent thymidylate synthase. NADP(H), nicotinamide adenine dinucleotide phosphate; CH2H4folate, N5,N10
methylenetetrahydrofolate; H4folate, tetrahydrofolate. R′ = (p-aminobenzoyl)glutamate and R″ = adenosine-5′-pyrophosphate-ribityl.
-
UV absorbance (at 264 and 450 nm). The concentration of riboflavin for
(ESI-MS) analysis was performed on a Waters Q-TOF mass spectrometer.
the reactions was determined by the absorbance at 445 nm
Flavin samples were directly infused for mass analysis in a 1:1 water:
acetonitrile solution. A À1 charge FAD ion was produced using collision
(ε = 12 200 cmÀ1 MÀ1
)
and that of final purified FAD at 450 nm
(ε = 11 300 cmÀ1 MÀ1). The concentration of FDTS for rapid-quenching energy of 4 eV. The expected shift in mass of 13C,15N-labeled flavins
experiments was determined by the 454-nm absorbance of bound was confirmed by comparison to the mass of unlabeled commercial
FAD (ε = 11 300 cmÀ1 MÀ1). Electron-spray ionization mass spectrometry
FAD standard, under identical analysis conditions.
www.jlcr.org
Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 370–375

T. V. Mishanina and A. Kohen
Purification of synthesized labeled flavins
The final synthesis mixture was passed through Amicon® Ultra
centrifugal filter (10 000 MWCO) to remove proteins and applied to
an HPLC semi-preparative reverse phase Supelco column (Discovery
series 250 mm × 10 mm, 5 μm) at a flow rate of 3.2 mL/min. Elution of
the flavins was monitored by 450-nm absorbance. Eluent containing
FAD (~15 min) was collected, purged with argon to remove methanol,
frozen and lyophilized to dryness. Care was taken to minimize exposure
of the flavin to light.
ApoFDTS preparation and reconstitution with labeled FAD
Recombinant FDTS from T. maritima was prepared as described
elsewhere.31 The enzyme was purified with a tightly bound FAD. To
remove this native FAD from the enzyme, sodium chloride solid was
added to the solution of FDTS to a final concentration of 30% w/v. This
enzyme solution was warmed to 40 °C and gently inverted to dissolve
NaCl solid. The aggregated protein was pelleted by centrifugation at
13 000 rpm and 4 °C, and washed with 30% w/v NaCl solution until
the pellet was visibly white, hence lacking bound FAD. This apoFDTS
was re-suspended in 50 mM Tris buffer, pH 8.0, 1 mM EDTA and washed
with Tris buffer by filtration to remove excess NaCl. ApoFDTS was then
incubated with labeled FAD at 1:1 concentration ratio overnight.
Binding of FAD to FDTS was corroborated by the shift of flavin λmax
from 450 nm (free FAD) to 454 nm (FDTS-bound FAD). This
reconstituted FDTS was washed with Tris buffer to remove any
unbound FAD, until the 280:454-nm absorbance ratio, indicative of
protein and flavin content in the solution, was ~6. With radiolabeled
FAD-FDTS, the amount of enzyme-bound 3H-labeled FAD was
determined by liquid-scintillation counting. The ability of the
reconstituted enzyme to convert dUMP to dTMP was always tested
prior to quenching experiments, and no loss in this thymidylate
synthase activity was ever observed upon reconstitution.
Figure 4. Negative-ion ESI-MS spectra of unlabeled FAD standard (A), purified 2
(B) and 4 (C). Note the expected mass shift of +15 for 2 and +6 for 4. Ions in m/z
800 region are because of formation of sodium and potassium adducts.
Base-quenching of labeled FAD-FDTS reactions
Rapid-quenching experiments with flavin-labeled FDTS were carried out
according to the published procedure,30 except with 1 M NaOH as the
reaction quencher. Reaction time points containing maximal
accumulation of the base-trapped intermediate were analyzed by
HPLC-LSC (radioactive samples) or LCMS (stable-isotope labeled
samples). In the experiments with 3H-labeled FAD, [2-14C]dUMP substrate
was used to track the base-trapped intermediate in HPLC analysis.
Results and discussion
Figure 5. HPLC radiograms of reaction mixtures for the synthesis of 3H-labeled
FAD: 1 (top panel) and 3 (bottom panel). (A, C) Synthetic mixtures at t-zero. Note
rapid formation of FMN intermediate in (C). (B, D) Reaction mixtures after 24-h
incubation.
Synthesis of labeled flavin nucleotides
In our excursion into the synthesis of labeled flavins, we were
mostly interested in labeled FAD, with purpose of employing
it in mechanistic studies of flavin-dependent thymidylate
synthase. Adenyl- and isoalloxazine-labeled FAD molecules evidenced by the radiogram in Figure 5D for instance (the
were each synthesized from the labeled ATP and riboflavin, tritiated peak at ~19 min can be attributed to unreacted
respectively. The incorporation of the isotopic labels was riboflavin). Large excess of ATP over riboflavin (at least 20-fold)
verified by MS for 13C,15N-labeled flavins (Figure 4), or was a key to complete conversion to FAD product. Amounts of
scintillation counting for tritiated compounds. The purity of ATP stoichiometric to riboflavin, on the other hand, halted
flavins was confirmed by direct infusion into MS (for stable reaction at FMN stage—conditions that may be used in the
isotopic labelling) or re-injection on HPLC with FSA detection synthesis of labeled FMN. FADs with all four isotopic-labeling
(for radiolabeled flavins), and the identity of synthesized patterns illustrated in Figure 2B were synthesized and HPLC
compounds as FAD was corroborated by their uptake by purified, as described in Experimental section. The final isolated
apoFDTS to produce an active enzyme. Inclusion of ADP → ATP FAD yields after HPLC purification were as follows: 40% for
recycling system (phosphocreatine/creatine phosphate kinase) compound 1 (radiochemical yield ~3% because of large
significantly improved enzymatic FAD yields, reaching >95% dilution of 3H-ATP with unlabeled ATP, see Experimental
conversion of riboflavin to FAD after 24-h reactions, as section); 47% for compound 2; 66% for compound
3
J. Label Compd. Radiopharm 2015, 58 370–375
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

T. V. Mishanina and A. Kohen
Demonstration of the utility of labeled FAD cofactors in
mechanistic studies of flavin-dependent thymidylate
synthase
Our rapid acid-quench of FDTS reactions produced a trapped
intermediate derivative, 5-hydroxymethyl-dUMP.30 In a base-
quenching experiment, on the other hand, a completely
different species is trapped. To test whether the base-trapped
intermediate contained any component of the enzyme-bound
FAD, we reconstituted the enzyme with isotopically labeled
FAD, using all four labeling patterns presented in Figure 2B. No
shift in mass of the trapped intermediate was observed with
compound 2, and no radioactivity was found on the trapped
intermediate when using compound 1. However, FDTS
reconstituted with 3 produced a tritiated trapped-intermediate
species (Figure 6), and with 4 a trapped species 2 Da heavier
than with unlabeled enzyme (Figure 7). These findings indicate
that rings A and B of FAD cofactor (Figure 1), but not the rest
of the isoalloxazine nucleus or the adenyl tail, are included in
the base-trapped intermediate. This is highly unusual and of
high potential impact on our understanding of FDTS mechanism,
because no covalent involvement of the flavin has ever been
proposed for FDTS.28,30 The efforts are underway to elucidate
the full structure of this base-trapped intermediate.
Figure 6. HPLC radiogram for the reaction of 3-reconstituted FDTS with [2-14C]
dUMP quenched at 1 s with 1 M NaOH. Radioactive counts from 14C (top) and 3H
(bottom) were determined by liquid-scintillation counting, which better separates
the β-spectra of these isotopes but results in a lower elution-time resolution relative
to FSA (Figure 5). The base-trapped intermediate clearly contains the 3H-labeled
isoalloxazine portion of FAD.
Conclusions
In light of their utility, efficient synthetic routes to the iso-
topically labeled flavins and analogues are of great interest.
We report the successful synthesis of four different isotopically
labeled FAD cofactors. Our synthetic route utilizes a bifunctional
FAD synthetase, which offers control over the final flavin
nucleotide product (FMN vs. FAD). The synthetic procedure
described here can be easily modified to convert riboflavin
analogues into their nucleotide forms.
To demonstrate their applicability to mechanistic problems,
we incorporated labeled FADs into flavin-dependent
thymidylate synthase and followed the integration of isotopes
into the unidentified base-trapped intermediate in the FDTS-
catalyzed reaction. The synthesized FAD cofactors provided
critical information about the FDTS mechanism which otherwise
would be challenging to obtain, i.e. that only two rings of the
isoalloxazine are included in the base-trapped intermediate,
while the rest of the isoalloxazine and adenyl portions are not.
Acknowledgements
This work was supported by NIH R01 GM065368 and NSF CHE
1149023 to AK, and the Iowa Center for Biocatalysis and
Bioprocessing to TVM, associated with NIH Predoctoral Training
Program in Biotechnology, Grant T32 GM008365.
Figure 7. Negative-ion ESI-MS spectra of the trapped intermediate in the
reactions with unlabeled FAD-FDTS (A) and 4-reconstituted FDTS (B). Note the
+2 Da shift in the mass of the trapped species and its sodium adducts upon
labeling of the enzyme-bound flavin cofactor (B). Potassium adducts observed in
panel A are because of trace amounts of potassium in the buffer used in
purification of the trapped intermediate (not used in purification of the compound
in panel B).
Conflict of interest
The authors did not report any conflict of interest.
References
[1] V. Massey, Biochem. Soc. Trans. 2000, 28, 283.
[2] R. Teufel, A. Miyanaga, Q. Michaudel, F. Stull, G. Louie, J. P. Noel, P. S.
Baran, B. Palfey, B. S. Moore, Nature 2013, 503, 552.
[3] M. E. Taga, N. A. Larsen, A. R. Howard-Jones, C. T. Walsh, G. C. Walker,
Nature 2007, 446, 449.
(radiochemical yield 40%) and 67% for compound 4. Specific
activities of the tritiated FAD were 0.245 and 0.9 Ci/mmol, for
compounds 1 and 3 respectively.
www.jlcr.org
Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 370–375

T. V. Mishanina and A. Kohen
[4] A. Haigney, A. Lukacs, R.-K. Zhao, A. L. Stelling, R. Brust, R.-R. Kim, M.
Kondo, I. Clark, M. Towrie, G. M. Greetham, B. Illarionov, A. Bacher, W.
Romisch-Margl, M. Fischer, S. R. Meech, P. J. Tonge, Biochemistry
2011, 50, 1321.
[5] J. Mehlhorn, H. Steinocher, S. Beck, J. T. M. Kennis, P. Hegemann, T.
Mathes, PLoS One 2013, 8, e79006.
[6] W. Rhead, V. Roettger, T. Marshali, B. Amendt, Pediatr. Res. 1993,
33, 129.
[7] M. Barile, T. A. Giancaspero, C. Brizio, C. Panebianco, C. Indiveri, M.
Galluccio, L. Vergani, I. Eberini, E. Gianazza, Curr. Pharm. Des. 2013,
19, 2649.
[16] G. Scola-Nagelschneider, P. Hemmerich, Eur. J. Biochem. 1976,
66, 567.
[17] P. Nielsen, P. Rauschenbach, A. Bacher, Methods Enzymol. 1986,
122, 209.
[18] A. W. Schrecker, A. Kornberg, J. Biol. Chem. 1950, 182, 795.
[19] R. Spencer, J. Fisher, C. Walsh, Biochemistry 1976, 15, 1043.
[20] S. M. H. Christie, G. W. Kenner, A. R. Todd, J. Chem. Soc. 1954, 46, 46.
[21] C. DeLuca, N. O. Kaplan, J. Biol. Chem. 1956, 223, 569.
[22] F. M. Huennekens, G. L. Kilgour, J. Am. Chem. Soc. 1955, 77, 6716.
[23] I. Efimov,V.Kuusk,X.Zhang,W.S.McIntire,Biochemistry1998,37,9716.
[24] H. Myllykallio, G. Lipowski, D. Leduc, J. Filee, P. Forterre, U. Liebl,
Science 2002, 297, 105.
[8] S. Ghisla, V. Massey, Biochem. J. 1986, 239, 1.
[9] D. P. Ballou, V. Massey, Biochemistry 1997, 36, 15713.
[25] Centers for Disease Control and Prevention. www.cdc.gov
[10] P. Chaiyen, J. Sucharitakul, J. Svasti, B. Entsch, V. Massey, D. P. Ballou, [26] D. Leduc, S. Graziani, L. Meslet-Cladiere, A. Sodolescu, U. Liebl, H.
Biochemistry 2004, 43, 3933.
[11] D. C. Haines, I. F. Sevrioukova, J. A. Peterson, Biochemistry
2000, 39, 9419.
Myllykallio, Biochem. Soc. Trans. 2004, 32, 231.
[27] H. Myllykallio, D. Leduc, J. Filee, U. Liebl, Trends Microbiol. 2003,
11, 220.
[12] A. Lukacs, A. Haigney, R. Brust, R.-K. Zhao, A. L. Stelling, I. P. Clark, M. [28] E. M. Koehn, T. Fleischmann, J. A. Conrad, B. A. Palfey, S. A. Lesley, I. I.
Towrie, G. M. Greetham, S. R. Meech, P. J. Tonge, J. Am. Chem. Soc.
2012, 133, 16893.
[13] Z. Liu, C. Tan, X. Guo, Y.-T. Kao, J. Li, L. Wang, A. Sancar, D. Zhong,
Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 14831.
Mathews, A. Kohen, Nature 2009, 458, 919.
[29] T. V. Mishanina, E. M. Koehn, A. Kohen, Bioorg. Chem. 2011, 43, 37.
[30] T. V. Mishanina, E. M. Koehn, J. A. Conrad, B. A. Palfey, S. A. Lesley, A.
Kohen, J. Am. Chem. Soc. 2012, 134, 4442.
[14] Z. Liu, X. Guo, C. Tan, J. Li, Y.-T. Kao, L. Wang, A. Sancar, D. Zhong,
J. Am. Chem. Soc. 2012, 134, 8104.
[15] L. A. Flexner, W. G. Farkas, Chem. Abstr. 1953, 47, 8781a.
[31] T. V. Mishanina, J. M. Corcoran, A. Kohen, J. Am. Chem. Soc. 2014,
136, 10597.
[32] M. M. Bradford, Anal. Biochem. 1976, 72, 248.
J. Label Compd. Radiopharm 2015, 58 370–375
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org