Pyruvate is the source of the two carbons that are required for formation of the imidazoline ring of 4-demethylwyosine

Article abstract of DOI:10.1021/bi2015053

TYW1 catalyzes the condensation of N-methylguanosine with two carbon atoms from an unknown second substrate to form 4-demethylwyosine, which is a common intermediate in the biosynthesis of all of the hypermodified RNA bases related to wybutosine found in eukaryal and archaeal tRNA^Phe. Of the potential substrates examined, only incubation with pyruvate resulted in formation of 4-demethylwyosine. Moreover, incubation with C1, C2, C3, or C1,2,3-¹³C-labeled pyruvate showed that C2 and C3 are incorporated while C1 is not. The mechanistic implications of these results are discussed in the context of the structure of TYW1.

Full text of DOI:10.1021/bi2015053

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Pyruvate Is the Source of the Two Carbons That Are Required for
Formation of the Imidazoline Ring of 4-Demethylwyosine
Anthony P. Young and Vahe Bandarian*
Department of Chemistry and Biochemistry, University of Arizona, 1041 East Lowell Street, Tucson, Arizona 85721-0088,
United States
S
* Supporting Information
We have established a biochemical assay to study the source
ABSTRACT: TYW1 catalyzes the condensation of N-
methylguanosine with two carbon atoms from an un-
known second substrate to form 4-demethylwyosine, which
is a common intermediate in the biosynthesis of all of the
hypermodified RNA bases related to wybutosine found in
eukaryal and archaeal tRNAPhe. Of the potential substrates
examined, only incubation with pyruvate resulted in for-
mation of 4-demethylwyosine. Moreover, incubation with
C1, C2, C3, or C1,2,3-13C-labeled pyruvate showed that
C2 and C3 are incorporated while C1 is not. The mech-
anistic implications of these results are discussed in the
context of the structure of TYW1.
of the two-carbon moiety that is required for the conversion of
m1G to imG-14. For ease of purification and stability, all of our
experiments were conducted with the Methanococcus jannaschii
homologues of TRM5 and TYW1. The biochemical function of
TRM5, conversion of G to m1G, has been documented;8
therefore, TRM5 could be utilized to generate m1G-containing
tRNAPhe in situ.
The recombinant M. jannaschii homologue of TYW1 was
purified anaerobically and reconstituted with Fe and S (see the
Supporting Information). The reconstituted protein was brown
and had an absorbance spectrum that displayed a characteristic
shoulder at 400 nm (Figure S3 of the Supporting Information).
The m1G-containing tRNAPhe (MJ_t16) was prepared by in
vitro runoff transcription (see the Supporting Information).
The assays were conducted anaerobically in the presence of
tRNAPhe, TYW1, TRM5, and SAM. The reaction mixtures
contained all components that have been shown to be required
for activity of TRM5 (SAM and Mg2+).8 In addition, sodium
dithionite was included to reduce the radical SAM cluster of
TYW1. Incubations were conducted for 12 h at 60 °C, fol-
lowing which reactions were quenched and the RNA was
extracted digested to nucleosides and analyzed via LC−MS as
described previously.9 The modified bases have distinct reten-
tion times and can be detected readily in the extracted ion chro-
matograms at m/z 298 (m1G) or 322 (imG-14); these correspond
to the [M + H+] ions of m1G and imG-14, respectively.
Four compounds, acetyl-CoA, acetyl phosphate, phosphoenol-
pyruvate, and pyruvate, were tested as possible two-carbon
sources. Of these, only incubation in the presence of pyruvate led
to a reduction of the m1G peak and the appearance of imG-14
(m/z 322) in the extracted ion chromatogram (Figure S4 of the
Supporting Information). We found that methyl viologen, while
not essential, stimulated activity; therefore, it was included in all
subsequent reactions. Control experiments established that imG-
14 does not form in the absence of dithionite, TYW1, or pyruvate
(Figure S4 of the Supporting Information). Control reactions
without SAM could not be conducted because it is a substrate for
TRM5, which is utilized for synthesis of the N-methylguanosine-
containing tRNAPhe in situ.
f the 151 modifications that have been documented in
ORNA, 92 occur in tRNA and many are conserved in all
domains of life.1 The biosynthetic pathways leading to these
modifications are often quite complex and ripe with novel
chemistries.
Wybutosine (yW) and its derivatives are found in position 37
of tRNA encoding Phe in eukaryotes and archaea.2 yW and its
derivatives are installed in a series of reactions (see Figure 1)
that all require S-adenosyl-L-methionine (SAM). The first step
of the pathway is methylation of N1 of G37 to generate
N-methylguanosine (m1G), which is converted to the tricyclic
ring of 4-demethylwyosine (imG-14) in a reaction catalyzed by
TYW1. imG-14 is converted to yW by the successive actions of
TYW2, TYW3, and TYW4. TRM5 and TYW1 homologues are
common to all organisms containing yW and yW derivatives.
Thus, imG-14 is a common intermediate to yW and yW
derivatives in all organisms that produce the hypermodified
base. Evidence of the biosynthetic pathway has accumulated
through gene knockout studies in yeast.3
TYW1 has been classified as a member of the radical SAM
superfamily4 on the basis of a conserved CxxxCxxC motif,
which provides three Cys thiolate ligands to form a catalytically
essential [4Fe-4S]+2/+ cluster. The cluster presumably binds
and reductively cleaves SAM to generate 5′-deoxyadenosyl
radical (dAdo•) for a radical-mediated transformation, as is the
case for other radical SAM proteins.5 In TYW1, the first step is
presumed to be abstraction of H from the methyl group of m1G
to initiate radical-mediated condensation with a two-carbon-
donating second substrate, leading to formation of imG-14. The
identity of the second substrate has remained elusive, thus
hampering mechanistic studies of TYW1, for which several
high-resolution structures are known.6,7
To gain additional insights into the mechanism by which
pyruvate, a three-carbon compound, can be the source of the
two carbons in the tricyclic system of yW, the experiments were
Received: September 27, 2011
Revised: October 23, 2011
Published: October 25, 2011
© 2011 American Chemical Society
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Figure 1. Biosynthetic pathways of yW. G at position 37 is transformed to imG-14 through the actions of TRM5 and TYW1, which is subsequently
converted to yW through the actions of TYW2, TYW3, and TYW4. The naming conventions used throughout this paper are for the free nucleoside.
repeated with several 13C-labeled analogues. Extracted ion
chromatographs at the m/z of imG-14 (m/z 322) and at +1 and
+2 are shown in Figure 2. In the presence of unlabeled
C3 of pyruvate are the source of the two carbon atoms that are
required for the synthesis of the tricyclic ring of yW. Note that
the smaller peaks in the chromatograms at +1 are from the nat-
ural abundance of isotopes of imG-14, which is expected to be
∼16% of the intensity of the [M + H+] peak in each instance.
We also conducted LC−MS/MS runs in which unlabeled or
singly labeled imG-14 (m/z 322 or 323, respectively) was
+
trapped and fragmented; in each case, the [MB2 ] ion corre-
sponding to the base carries the appropriate m/z, consistent
with previous imG-14 fragmentation studies.3,10
Interpretation of the pyruvate labeling results with TYW1
can be conducted in the context of the two structures and
mutagenesis results that are available in the literature.6,7 Six Cys
residues are conserved in TYW1, three of which are in the
radical SAM signature sequence (C62xxxC66xxC69, M. jannaschii
numbering). The thiolate side chains of these residues would be
expected to bind three of the metal ions in the [4Fe-4S] cluster;
the fourth iron is presumably coordinated to the α-amino and
α-carboxylate of SAM, as has been shown previously for other
radical SAM proteins.11 The radical SAM cluster is located on
one side of a positively charged putative active site cleft, where
tRNA is proposed to bind and flip the m1G precursor for
modification.7 The three additional conserved Cys residues
(Cys26, Cys39, and Cys52) are located adjacent to the sub-
strate binding site on the opposite side of the cleft from the
SAM binding cluster. It has been proposed that these Cys re-
sidues could also form a [4Fe-4S] cluster. A conserved Lys
residue (K41) adjacent to the second set of conserved Cys
residues is also required for activity on the basis of in vivo
studies.6 When one considers where pyruvate would bind, it
seems reasonable to propose that the pyruvate binding site is
on the same side as the Lys, with which it may form a Schiff
base to facilitate the chemistry.
Our working model for the mechanism of TYW1, shown in
Figure 3, is as follows. We propose that the conserved Lys
residue forms a Schiff base with pyruvate. Reductive cleavage of
SAM leads to formation of dAdo•, which either directly or
perhaps through a protein side chain propagates the radical to
the m1G, generating a substrate radical. Addition of the radical
to C2 of pyruvate followed by homolytic scission of the C1−C2
bond would generate an intermediate, which through
transimination and subsequent deprotonation forms imG-14.
Decarboxylations, such as that proposed here, have
been proposed in the reaction of pyruvate formate-lyase12
and coproporphyrinogen synthyase (HemN).13 The resulting
formyl radical can either acquire an H atom or undergo
reduction and protonation to produce formate. Alternatively,
oxidation of the formyl radical would generate CO2. At this
Figure 2. Extracted ion chromatograms showing incorporation of C2
and C3 of pyruvate into 4-demethylwyosine.
pyruvate, the extracted ion chromatograms show a peak at the
expected m/z value of 322. Interestingly, when the reaction was
conducted in the presence of [1-13C1]pyruvate, an identical set
of extracted ion chromatographs was obtained, indicating loss
of C1. When the assays were conducted with [2-13C1]- or
[3-13C1]pyruvate, however, the peak at m/z 322 disappeared
and a peak appeared in the extracted ions chromatogram at
m/z 323. This suggests that both C2 and C3 of pyruvate are
incorporated into the tricyclic ring of yW. Consistent with these
labeling patterns, when the reaction was conducted in the
presence of [1,2,3-13C3]pyruvate, only a +2 shift to m/z 324
was observed. These results clearly demonstrate that C2 and
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ABBREVIATIONS
■
yW, wybutosine; SAM, S-adenosyl-L-methionine; m1G,
N-methylguanosine; imG-14, 4-demethylwyosine; dAdo•,
5′-deoxyadenosyl radical.
REFERENCES
■
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(11) Vey, J. L., and Drennan, C. L. (2011) Structural insights into
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(12) Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., and Ganzler,
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Figure 3. Putative mechanism for the transformation of N-
methylguanosine to 4-demethylwyosine.
point, given the uncertainty about the involvement of enzyme-
based radicals and the nature of the structure formed by the
second set of conserved Cys residues, it is difficult to differen-
tiate among these many possibilities. We note that while a Lys
residue would be desirable, the transformations proposed in
Figure 3 could also occur with pyruvate alone; however, a Schiff
base would provide an attractive electron sink to stabilize the
intermediates. The M. jannaschii protein used in these studies
lacks a flavin mononucleotide binding domain that appends the
protein in higher organisms; its absence from archaeal proteins
may indicate that it has an alternative mechanism for cluster
reduction.
In summary, we have identified pyruvate as the second
substrate for TYW1 in the production of imG-14 on the
pathway to yW. We have successfully reconstitued activity in
vitro using isotopically labeled pyruvate. The isotope labeling
patterns unambiguously show that C2 and C3 of pyruvate are
incorporated into the tricyclic base, whereas C1 is lost. These
observations, when taken with the X-ray crystal structures,
conserved residues, and mutagenesis data in the literature,3,6,7,14
provide support for the model proposed here, which will be
useful in directing future mechanistic studies of the fascinating
transformation catalyzed by TYW1.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed methods and materials. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Department of Chemistry and Biochemistry, University of
Arizona, 1041 E. Lowell St., BioSciWest 540, Tucson, AZ
85721-0088. Telephone: (520) 626-0389. Fax: (520) 626-9204.
E-mail: vahe@email.arizona.edu.
Funding
We are grateful for support from National Institutes of Health
Grant GM 72623 and a Career Award in Biomedical Sciences
from the Burroughs Wellcome Fund.
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