Evolution of eukaryal tRNA-guanine transglycosylase: Insight gained from the heterocyclic substrate recognition by the wild-type and mutant human and Escherichia coli tRNA-guanine transglycosylases

Article abstract of DOI:10.1093/nar/gkq1188

The enzyme tRNA-guanine transglycosylase (TGT) is involved in the queuosine modification of tRNAs in eukarya and eubacteria and in the archaeosine modification of tRNAs in archaea. However, the different classes of TGTs utilize different heterocyclic substrates (and tRNA in the case of archaea). Based on the X-ray structural analyses, an earlier study [Stengl et al. (2005) Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. Chembiochem, 6, 1926-1939] has made a compelling case for the divergent evolution of the eubacterial and archaeal TGTs. The X-ray structure of the eukaryal class of TGTs is not known. We performed sequence homology and phylogenetic analyses, and carried out enzyme kinetics studies with the wild-type and mutant TGTs from Escherichia coli and human using various heterocyclic substrates that we synthesized. Observations with the Cys145Val (E. coli) and the corresponding Val161Cys (human) TGTs are consistent with the idea that the Cys145 evolved in eubacterial TGTs to recognize preQ1 but not queuine, whereas the eukaryal equivalent, Val161, evolved for increased recognition of queuine and a concomitantly decreased recognition of preQ1. Both the phylogenetic and kinetic analyses support the conclusion that all TGTs have divergently evolved to specifically recognize their cognate heterocyclic substrates.

Full text of DOI:10.1093/nar/gkq1188

2834–2844 Nucleic Acids Research, 2011, Vol. 39, No. 7
doi:10.1093/nar/gkq1188
Published online 3 December 2010
Evolution of eukaryal tRNA-guanine
transglycosylase: insight gained from the
heterocyclic substrate recognition by the
wild-type and mutant human and Escherichia coli
tRNA-guanine transglycosylases
Yi-Chen Chen, Allen F. Brooks, DeeAnne M. Goodenough-Lashua, Jeffrey D. Kittendorf,
Hollis D. Showalter and George A. Garcia*
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor,
MI 48109-1065, USA
Received October 20, 2010; Revised November 3, 2010; Accepted November 4, 2010
ABSTRACT
phylogenetic and kinetic analyses support the con-
clusion that all TGTs have divergently evolved to
specifically recognize their cognate heterocyclic
substrates.
The enzyme tRNA-guanine transglycosylase (TGT)
is involved in the queuosine modification of tRNAs
in eukarya and eubacteria and in the archaeosine
modification of tRNAs in archaea. However, the dif-
ferent classes of TGTs utilize different heterocyclic
substrates (and tRNA in the case of archaea). Based
on the X-ray structural analyses, an earlier study
[Stengl et al. (2005) Mechanism and substrate spe-
cificity of tRNA-guanine transglycosylases (TGTs):
tRNA-modifying enzymes from the three different
kingdoms of life share a common catalytic mechan-
ism. Chembiochem, 6, 1926–1939] has made a
compelling case for the divergent evolution of the
eubacterial and archaeal TGTs. The X-ray structure
of the eukaryal class of TGTs is not known. We
performed sequence homology and phylogen-
etic analyses, and carried out enzyme kinetics
studies with the wild-type and mutant TGTs
from Escherichia coli and human using various
heterocyclic substrates that we synthesized.
Observations with the Cys145Val (E. coli) and the
corresponding Val161Cys (human) TGTs are con-
sistent with the idea that the Cys145 evolved in eu-
bacterial TGTs to recognize preQ1 but not queuine,
whereas the eukaryal equivalent, Val161, evolved
for increased recognition of queuine and a concomi-
tantly decreased recognition of preQ1. Both the
INTRODUCTION
Transfer RNA-guanine transglycosylase (TGT, EC
2.4.2.29) is the key enzyme responsible for the queuine
[7-((4,
5-cis-dihydroxy-2-cyclopenten-1-yl)
amino]
methyl-7-deazaguanine (Scheme 1) modification of
tRNA in eukarya and eubacteria and archaeosine in
archaea (1). In eukarya and eubacteria, TGT incorporates
queuine or preQ1 (7-aminomethyl-7-deazaguanine;
Scheme 1), respectively, into the anticodon wobble
position (position 34) of tRNAs (2,3), while archaeal
TGT places another 7-deazaguanine analog, preQ0
(7-cyano-7-deazaguanine), into the position 15 (D loop)
of tRNAs (4).
The TGT reaction shares a common mechanism
through the replacement of
a genetically encoded
guanine, shown for the eubacterial TGT to occur via a
covalent TGT–RNA intermediate with ping-pong
kinetics (5–7). Also, crystallographic evidence reveals
that the N-terminal domains of eubacterial and archaeal
TGTs fold into an (b/a)8 TIM barrel, while a characteris-
tic zinc-binding motif is found in their C-terminal domains
(8,9). Romier et al. (8) observed a dimer interface when
the structure of the Zymomonas mobilis TGT was
determined. Later, co-crystallization of the enzyme with
*To whom correspondence should be addressed. Tel: +1 734 764 2202; Fax: +1 734 647 8430; Email: gagarcia@umich.edu
Present addresses:
DeeAnne M. Goodenough-Lashua, Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556-5670, USA.
Jeffrey D. Kittendorf, Alluvium Biosciences, Ann Arbor, MI, USA.
ß The Author(s) 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acids Research, 2011, Vol. 39, No. 7 2835
Scheme 1. Synthesis of 1H- and 3H-labeled PreQ1 and Queuine: (a) trityl amine, Na2SO4, anhydrous THF, reflux, 6 h; (b) 2eq. NaBH4 or NaB3H4,
THF, 0–25ꢀC, 3 h, 79% two steps; (c) 1.25 M methanolic HCl, reflux, 2 h, 89%; (d) (3S,4R,5S)-3-amino-4,5-isopropylidenedioxy-cyclopentene,
Na2SO4, MeOH, 25ꢀC, 6 h (32); (e) 2eq. NaBH4 or NaB3H4, MeOH, 0–25ꢀC, 4 h, 99% two steps (32); (f) 1.25 M methanolic HCl, 25ꢀC, 2.5 h,
87% (32).
an anticodon stem–loop confirmed a homodimer forma-
tion upon tRNA binding (10). The archaeal TGT has also
been shown to contain two monomers per asymmetric unit
and the two subunits were suggested to interact tightly
through the zinc-binding domain (9). The most interesting
subunit structure was found in eukarya. Although lacking
a crystal structure, the eukaryal TGT has been proposed
for almost four decades to be a heterodimer (11) based
upon biochemical and kinetic characterizations.
Although there have been discrepancies regarding the
reported size and composition of the subunits (11–14), it
is now clear that the eukaryal TGT is composed of
queuine tRNA-ribosyltransferase (QTRT1) and QTRT
domain-containing 1 (QTRTD1), which are homologous
subunits of 44 and 46.7 kDa, respectively (15,16).
QTRTD1 has been proposed to be the queuine salvage
enzyme that liberates free queuine from QMP (16).
An argument has been made that queuosine modifica-
tion in eubacteria and eukarya may have resulted from
convergent evolution based on the dramatic differences
between their queuosine modification systems (e.g.
eukarya do not synthesize queuine while eubacteria do,
and eukarya transport and salvage queuine while
eubacteria do not) (17). At that time, the quaternary struc-
ture of the eukaryal TGT was thought to be different from
that of the eubacterial TGT, as described above.
Subsequently, based upon careful analyses of the X-ray
crystal structures of eubacterial and archaeal TGTs,
Klebe et al. (18) have presented a compelling case for
the divergent evolution of TGT. Their evidence includes
the close overall structural homology and the absolute
conservation of zinc-binding and key active-site residues.
They also present a cogent discussion of changes in key
amino acids in the active site that are responsible for the
differential heterocyclic substrate recognition between the
eubacterial (preQ1) and archaeal (preQ0) TGTs. However,
in the absence of an X-ray crystal structure and any
detailed biochemical evidence, extension of the divergent
evolution concept to the eukaryal TGT could only be
inferred from sequence homologies.
To confirm the divergent evolution model for TGT,
we report further sequence homology and phylogenetic
analyses, the results of which are consistent with divergent
evolution. To provide experimental evidence for the diver-
gent evolution of TGT in eukaryotes, queuine and preQ1
incorporation studies were performed with wild-type and
mutant human and Escherichia coli tRNA-guanine
transglycosylases. Enzymological studies of mutants of
Cys145 (E. coli TGT) and the corresponding Val161
(human TGT) are consistent with the concept that this
residue, in particular, has evolved to enhance recognition
of preQ1 in eubacteria and to decrease recognition of
preQ1, concomitant with increased recognition of
queuine, in eukarya. These phylogenetic analyses and ex-
perimental results support the conclusion that all TGTs
have divergently evolved to specifically recognize their
cognate heterocyclic substrates, while minimizing recogni-
tion of non-cognate ones.
MATERIALS AND METHODS
Reagents
Unless otherwise specified, all reagents were ordered
from Sigma-Aldrich. DNA oligonucleotides, agarose,
dithiothreitol (DTT) and DNA ladders were ordered

2836 Nucleic Acids Research, 2011, Vol. 39, No. 7
Aspartates 89, 143, 264
Cysteines 145, 302, 304, 307
Histidine 333
E.coli
...GPILTDSGGFQ...IVMIFDECTPY...GIDMFDCVMPTR...PLDPECDCYTCRNYSR...RLNTIHNLRYY...
Z.mobilis
S.flexneri
B.subtilis
A.aeolicus
...RPILTDSGGYQ...IVMAFDECTPY...GIDMFDCVLPTR...PLDSECHCAVCQKWSR...MLMTEHNIAFY...
...GPILTDSGGFQ...IVMIFDECTPY...GIDMFDCVMPTR...PLDPECDCYTCRNYSR...RLNTIHNLRYY...
...RAILTDSGGFQ...IMMAFDECPPY...GVDMFDCVLPTR...PIDEECDCYTCKNYTR...RLTTYHNLHFL...
...KPILTDSGGYQ...IMMPLDECVEY...GVDMFDCVVPTR...PLDPECDCYTCRNFSK...VLNTIHNLRFY...
M.acetivorans ...GPIMTDSGSFQ...IIVPLDIPTPP...GCDLFDSAAYAL...P----CSCPVCSKYTA...ELLGKHNLYAT...
M.mazei
...GPIMTDSGSFQ...IIVPLDIPTPP...GCDLFDSAAYAL...P----CSCPVCSRYTA...ELLGRHNLYAT...
...GAIMTDSGSFQ...IATPVDIPTPP...GCDLFDSAAYAL...P----CTCPICTEYSP...QLLAEHNLHVT...
...MPVMTDSGSYQ...IIVPLDIPTPP...GCDLFDSAAYAL...P----CKCPVCSNHDP...RLIAEHNLYVS...
...GIIEVDSGSFQ...IGTFLDIPTPP...GVDLFDSASYAL...P----CSCPVCSKYTP...RLLALHNLWVI...
...RSILTDSGGFQ...IMMQLDHVIHV...GADMFDCVYPTR...PIDKKCECNTCKNYTR...HLVSVHNIKHQ...
H.volcanii
A.fulgidus
P.horikoshii
C.elegans
D.melanogaster ...RAILTDSGGFQ...IMMQLDDVVKT...GIDMFDCVFPTR...PIDKDCDCSTCRRYTR...SLLSIHNVAYQ...
M.musculus
R.norvegicus
H.sapien
...HNLLTDSGGFQ...IIMQLDHVVSS...GCDMFDCVYPTR...PINPECPCPTCQTHSR...HHLTVHNIAYQ...
...HNLLTDSGGFQ...IIMQLDDVVSS...GCDMFDCVYPTR...PINPECPCATCQTHSR...HHLTVHNIAYQ...
...HNLLTDSGGFQ...IIMQLDDVVSS...GCDMFDCVFPTR...PIDPECTCPTCQKHSR...HHLTVHNIAYQ...
Figure 1. Selected regions of a sequence homology analysis of TGTs across the three kingdoms of life. A sequence alignment of representative TGTs
from eubacteria, archaea and eukarya highlighting the conservation of aspartates 89, 143 and 264; cysteines 145, 302, 304 and 307; and histidine 333
(E. coli numbering). Dots indicate regions intentionally deleted for this figure. Dashes indicate gaps in the sequence alignment. The full alignment (see
Supplementary Figure S17) was generated using PileUP in the SeqWeb software package (Accelerys). The conserved amino acids are colored-coded
red.
from Invitrogen. The human tRNATyr gene was
synthesized by The Midland Certified Reagent
Company. All restriction enzymes and VentÕ DNA poly-
merase were ordered from New England Biolabs. The
ribonucleic acid triphosphates (NTPs), pyrophosphatase
and kanamycin sulfate were ordered from Roche
Applied Sciences. The deoxyribonucleic acid triphos-
phates (dNTPs) were ordered from Promega.
ScriptguardTM RNase Inhibitor was ordered from
Epicentre. Epicurian coliÕ XL2-Blue ultracompetent
cells were ordered from Agilent Technologies, E. coli
TG2 and BL21 (DE3) cells were from laboratory stocks.
HisꢁBind resin and lysonase bioprocessing reagent were
purchased from Novagen. The QIAPrepÕ Spin Miniprep
and Maxiprep Kits were ordered from Qiagen. Precast
PhastGels and SDS buffer strips were from VWR.
Bradford reagent was from Bio-Rad. Whatman GF/C
Glass Microfibre Filters, Amicon Ultra Centrifugal
Filter Devices, carbenicillin and all bacterial media com-
ponents were ordered from Fisher. [8-14C]-Guanine
(50–60 mCi/mmol) was ordered from Moravek
Biochemicals and the tritiation of [3H]-preQ1 and [3H]-
queuine was also performed by Moravek Biochemicals
(see below). T7 RNA polymerase was isolated from
E. coli BL21 (DE3) pLysS cells harboring the plasmid
pRC9 via minor modifications of the procedure described
in the literature (19). The human TGT and tRNATyr
(human and E. coli) were generated as previously
described (16,20).
Methanosarcina acetivorans, NP_619281; M. mazei,
NP_633125; Haloferax volcanii, BAB40327;
Archeoglobus. fulgidus, NP_070314; Pyrococcus horikoshii,
NP_143020; Caenorhabditis elegans, NP_502268;
Drosophila melanogaster, NP_608585; Mus musculus,
NP_068688; Rattus norvegicus, NP_071586; Homosapien,
AAG60033). Sequence alignment was performed using the
PileUP alignment program found in the SeqWeb software
package (Accelrys). Selected regions highlighting the con-
servation of aspartates 89, 143 and 264; cysteines 145, 302,
304 and 307; and histidine 333 (E. coli numbering) are
shown in Figure 1. The full alignment can be found in
Supplemetary Figure S12.
Phylogenetic tree for TGTs across the three kingdoms
Amino acid sequences for eubacterial and archaeal TGT
and eukaryal QTRT1 and QTRTD1 were manually ex-
tracted from the NCBI protein sequence database. A
very large number of sequences (approximately 6000)
were initially found. In the case of archaeal TGTs, these
were sorted by phylogenetic tree and duplicates were dis-
carded. Where a number of examples were present within
each subgroup, only 1–2 were selected for inclusion in our
study as we wanted the sample to be a diverse representa-
tion and not to be biased towards any one subgroup. A
large number of protein sequences were annotated as
‘PUA domain containing’; however, only those clearly
annotated as tRNA transglycosylase or ribosyltransferase
(e.g. queuine, guanine, 7-cyano-preQ1 or archaeosine)
were included to avoid possible confusion with pseudo-
uridine synthases and others. Due to the large number
of subgroups for the eubacterial TGTs, the sequences
were sorted by the highest level of identified organisms
listed in the database; however, only one example per
genus was chosen to minimize bias.
Syntheses of [3H]-PreQ1 (2) and [3H]-Queuine (4)
The detailed procedures for these syntheses can be found
in the Supplemetary data.
Multiple sequence alignment of representative TGTs
across the three kingdoms
A multiple sequence alignment was then performed with
Clustal W. A number of sequences (36) were found to be
suspect. Reexamination of the NCBI database allowed us
to correct 30 of these sequences. The six sequences that
were deleted were found to not align with (or were
missing) the conserved active-site aspartates and
The protein sequences of 15 TGTs, spanning the three
phylogenic domains, were retrieved using the following
accession numbers (E. coli, AAA24667; Z. mobilis,
T46898; Shigella flexneri, NP_706294; Bacillus subtilis,
NP_390649;
Aquifex
aeolicus,
NP_213895;

Nucleic Acids Research, 2011, Vol. 39, No. 7 2837
mutagenic primers: 50-CATCATCATGCAGCTGGAC
GACTGTGTTAGCAGTACTGTGACTGGGCC-30 and
50-GGCCCAGTCACAGTACTGCTAACACAGTCGT
CCAGCTGCATGATGATG-30. The Val161Cys mutant
was then prepared in the same fashion as the wild-type.
Construction of E. coli TGT Cys145 mutants
The Cys145Ala plasmid was previously generated (23) by
a combined polymerase chain reaction (PCR)-ligase chain
reaction (24). The Cys145Ser plasmid was generated by
site-directed mutagenesis using pTGT5 as a template
(25) and the following mutagenic primers 50-GCAGGAT
ACGGCGTAGACTCATCAAAGATCATGACG-30 and
50-CGTCATGATCTTTGATGAGTCTACGCCGTATC
CTGC-30. The Cys145Asp plasmid was generated as
described for Cys145Ser with the following changes: mu-
tagenic primers (50-GCAGGATACGGCGTGTCTTCAT
CAAAGATCATGACG-30 and 50-CGTCATGATCTTT
GATGAAGACACGCCGTATCCTGC-30), the total
volume of the PCR reaction was 25 ml and the primer con-
centration was reduced to 0.6 mM. The Cys145Asp PCR
product formed DNase sensitive aggregates that were
immobile on an agarose gel, and remained after the
wild-type plasmid was eliminated by Dpn I digestion.
Therefore, the PCR product was diluted 5-fold prior to
transformation into competent cells. All resulting
plasmids were isolated via Qiagen miniprep, and the tgt
mutant genes were confirmed by DNA sequencing
(University of Michigan DNA Sequencing Core
Facilities). To generate the plasmids of polyhistidine-
tagged E. coli mutants, the tgt mutant genes were
subcloned into vector pET-28aKan following general la-
boratory protocols. The plasmid of polyhistidine-tagged
Cys145Val was directly generated using the plasmid of
polyhistidine-tagged Cys145Ala as a template and the fol-
lowing mutagenic primers: 50-CAGTCAGCAGGATACG
Figure 2. Phylogenetic tree for TGTs across the three Kingdoms. The
evolutionary history of the TGT enzymes was inferred via ‘maximum
likelihood’ analysis with the MEGA4 software package. Representative
TGT sequences spanning the three domains of life (13 eukaryal, 59
eubacterial and 72 archaeal) were globally aligned using Clustal W.
The alignment was subjected to 500 bootstrap replicates resulting in
the final consensus phylogenetic tree. This is an unrooted tree and the
branch lengths are proportional to the evolutionary distances between
nodes. Due to the number of sequences, a condensed version of the tree
is shown. The full tree can be seen in Supplementary Figure S13.
zinc-binding ligands (Figure 1) or were of lengths not con-
sistent with the other sequences. This data set contained
approximately 100 eubacterial sequences. Our initial tree
calculation suggested that the disproportionate number
of eubacterial sequences might be biasing the analysis.
We therefore pared down the number of eubacterial
sequences using the same criterion of maintaining
phyogenetic diversity. This yielded 13 eukaryal QTRT1,
72 archaeal TGT and 59 eubacterial TGT sequences. A
listing of these sequences can be found in Supplemetary
Table S1.
GCGTAACCTCATCAAAGATCATGACG-30
and
50-CGTCATGATCTTTGATGAGGTTACGCCGTATC
CTGCTGACTG-30.
Expression and purification of E. coli TGT and Cys145
mutants
The wild-type E. coli TGT and Cys145 mutants were
prepared following the procedure established by Chong
and Garcia (25), with minor modifications. The final
enzyme concentrations were determined with the
Bio-Rad Protein Assay Kit using BSA as the standard.
The proteins were stored in liquid N2 and utilized to de-
termine guanine and tRNA kinetic parameters. The
wild-type polyhistidine-tagged E. coli TGT was prepared
as described previously (20), and the polyhistidine-tagged
E. coli TGT mutants were expressed following an
auto-induction procedure (26). The final concentration
of each protein was determined as described above and
the proteins were stored in liquid N2 and utilized to deter-
mine preQ1 kinetic parameters. Note that we have previ-
ously determined that the polyhistidine tag has no effect
on the TGT reaction or kinetics (27).
The evolutionary history of the TGT enzymes was then
inferred via ‘maximum likelihood’ analysis using the
MEGA4 software package (21,22). The Clustal W align-
ment was subjected to 500 bootstrap replicates resulting in
the final consensus phylogenetic tree. Due to the number of
sequences, a condensed version of the tree is shown
in Figure 2. The full tree can be seen in Supplemetary
Figure S13.
Construction, expression and purification of human TGT
and Val161Cys mutant
The wild-type human TGT was prepared as described pre-
viously (16). The Val161Cys mutant plasmid was generated
by QuikChangeTM site-directed mutagenesis (Stratagene)
using the wild-type plasmid as a template and the following

2838 Nucleic Acids Research, 2011, Vol. 39, No. 7
Activity screen and kinetic analyses
more harsh reaction conditions that would require
multiple chromatographic steps following incorporation
of label via [3H]-formaldehyde. The recent synthesis of
Klepper et al. (30) proceeds via readily available preQ0
(31), but the second stage reduction of the nitrile to the
aminomethylene group by hydrogenation proceeded only
in 10% yield and required tedious purification. Hence,
none of these procedures was deemed suitable for our
needs. This article reports an exceptionally short, and
high yielding synthesis of preQ1 (1) from readily available
precursors and its utilization in the synthesis of 3H-labeled
congener 2.
Our route is outlined in Scheme 1. Reaction of easily
derived aldehyde 5 (32) was examined with a number of
ammonia surrogates (benzyl amine, 4-methoxybenzyl
amine, 2,4-dimethoxybenzyl amine). While reductive
amination proceeded cleanly, subsequent global
deprotection under catalytic hydrogenolysis or standard
acid conditions did not proceed satisfactorily. Trityl
amine was next explored as a surrogate and was found
to be ideal. Thus, heating 5 with trityl amine in refluxing
THF cleanly formed stable imine 6 in essentially quanti-
tative yield. Reduction of 6 with two equivalents of
sodium borohydride then provided 7 in 79% yield follow-
ing filtration through a plug of silica gel. This was entered
directly into a global deprotection with methanolic HCl to
provide preQ1 (1), which precipiated out of solution as the
monohydrochloride salt in 89% yield. The exact same
process was then repeated by Moravek Biochemicals
using [3H]-sodium borohydride to incorporate the label.
The collected product 2 had a specific activity of
7.0 Ci/mmol, and showed chemical and radiochemical
purities of 99.5% and 96.1%, respectively, by HPLC
(Supplemetary Figures S7 and S8).
Heterocyclic base-exchange assays were conducted by
monitoring the incorporation of radiolabeled substrates,
[8-14C]-guanine, [3H]-preQ1 and [3H]-queuine into either
the human or E. coli tRNATyr using various TGT
samples. In brief, kinetic assays were set up under the
following conditions: tRNATyr (saturating concentrations
depending on the Km for each individual enzyme), individ-
ual radiolabeled base (various concentrations), TGT
(25, 50 or 100 nM depending on the concentration of het-
erocyclic base used) and HEPES reaction buffer (100 mM
HEPES, pH 7.3; 20 mM MgCl2; 5 mM DTT) to a final
volume of 400 ml. The studies were performed in triplicate.
All samples were incubated at 37ꢀC for purposes of equili-
bration before initiating the reaction with the addition of
TGT. Aliquots (70 ml) were removed every 2 min through-
out the 10-min time course and immediately quenched in
2.5 ml of 5% trichloroacetic acid (TCA) for 1 h before
filtering on glass-fiber filters. Each filter was washed with
three volumes of 5% TCA and a final wash of ethanol to
dry the filter. The samples were analyzed in a scintillation
counter (Beckman) for radioactive decay, where counts
were reported in DPM and later converted to picomoles
[8-14C]-guanine/[3H]-preQ1/[3H]-queuine by following the
general conversion between DPM and specific activity
(e.g., pmol = DPM ꢂ 0.0079, for the [8-14C]-guanine
stock with a specific activity of 57 mCi/mmol). To obtain
steady-state kinetic parameters, initial velocities of
guanine/preQ1/queuine incorporation were determined
by converting the slopes of these plots (pmol/min) to
units of per second (1/sec), taking into account the con-
centration of the enzyme and aliquot size. The individual
data points from each trial were averaged, and the
standard deviation was determined for each concentration
of either tRNATyr or heterocyclic base. The average data
points (with error bars representing their standard devi-
ations) were plotted. However, all of the individual data
points were fit via non-linear regression to the Michaelis–
Menten equation using KaleidaGraph (Abelbeck
Software).
Synthesis of [3H]-Queuine (4)
Our route to [3H]-queuine (4) is outlined in Scheme 1 and
follows directly from our recently published short,
high-yielding synthesis of queuine (3) (32). The collected
product 4 (by Moravek Biochemicals) had a specific
activity of 7.3 Ci/mmol and showed chemical and radio-
chemical purities of 94.1 and 99.4%, respectively, by
HPLC (Supplemetary Figures S9–11).
RESULTS
Phylogenetic tree for TGTs across the three kingdoms
Improved synthesis of PreQ1 (1) and application to
preparation of [3H] PreQ1 (2)
A complete search of the NCBI database for TGT se-
quences and subsequent paring down of some sequences
yielded a representative number of sequences that were
aligned via Clustal W. This alignment then was used for
a phylogenetic tree analysis using MEGA4. The tree
analysis (Figure 2) clearly demonstrates the relatedness
of the TGT sequences. Consistent with the simple
homology analysis, the archaeal sequences clearly group
distinctly from the others and the eukaryal sequences
group together, but closer to the eubacterial sequences.
In consideration of a suitable synthetic scheme to make
[3H]-preQ1 (2), a short, efficient route that could be easily
executed with simple reagents was desired. An examin-
ation of the literature for the synthesis of cold-labeled
compound 1 revealed three previous reports (28–30).
The original report of Ohgi et al. (28) provides 1 in
6.7% overall yield and requires 12 steps from a
non-commercial starting material. A second synthesis by
Akimoto et al. (29) is much shorter and proceeds via a
Mannich reaction to incorporate the aminomethylene side
chain of preQ1 regioselectively to the C-5 position of a
pyrrolopyrimidine precursor. Its main drawback from a
standpoint of radiochemical synthesis is the co-generation
of a small amount of C-6 substituted isomer and generally
Kinetics analysis of human TGT with respect to Queuine
and PreQ1
To investigate the heterocyclic substrate specificity of the
human TGT (SDS-PAGE in Supplemetary Figure S3A,

Nucleic Acids Research, 2011, Vol. 39, No. 7 2839
Table 1. Kinetic parameters for eukaryotic TGTs with heterocyclic
substrates
a,b
kcat
(10ꢄ3ꢁsꢄ1
a,b
Km
(mM)
a,b
kcat/Km
(10ꢄ3ꢁsꢄ1ꢁmMꢄ1
Enzyme
)
)
Human
Queuine
8.22 (0.20)
8.23 (0.71)
5.86 (0.10)
0.26 (0.03)
132 (28)
0.41 (0.03)
31.6 (3.4)
0.062 (0.014)
14.2 (0.9)
PreQ1
Guaninec
Rat liverd
Queuine
PreQ1
N.D.f
N.D.
N.D.
0.29g
2.1g
N.D.
N.D.
N.D.
Guanine
0.83g
Rabbit Erythrocytese
Guanine
N.D.
0.15g
N.D.
aStandard errors are shown in parentheses.
bKinetic parameters were calculated from the average of three replicate
determinations of initial velocity data.
cData from Chen et al. (16).
dData from Shindo-Okada et al. (3).
eData from Howes and Farkas (11).
fNot Determined.
gNo errors reported.
Table 2. PreQ1 kinetics for wild-type and position 145/161 mutant
TGTs
a,b
kcat
(10ꢄ3ꢁsꢄ1
a,b
Km
(mM)
a,b
kcat/Km
(10ꢄ3ꢁsꢄ1ꢁmMꢄ1
Enzyme
)
)
Escherichia coli
Wild-type
9.57 (0.24)
14.1 (0.7)
74.6 (3.4)
23.7 (1.0)
N.D.A.c
0.05 (0.01)
2.76 (0.5)
43.4 (5.0)
123 (13.9)
N.D.A.c
191 (38.6)
5.10 (0.95)
1.72 (0.21)
0.19 (0.02)
N.D.A.c
Cys145Val
Cys145Ala
Cys145Ser
Cys145Asp.
Human
Val161Cys
4.64 (0.32)
22.3 (4.1)
0.21 (0.04)
aStandard errors are shown in parentheses.
bKinetic parameters were calculated from the average of three replicate
determinations of initial velocity data.
cNo detectable activity ꢅ100 mM preQ1.
Figure 3. Michaelis–Menten fits of (A) Queuine and (B) PreQ1 with
wild-type human TGT.
Kinetics analysis of E. coli TGT with respect to Guanine,
PreQ1 and Queuine
Similarly, the transglycosylase activity of the E. coli TGT
(SDS–PAGE in Supplemetary Figure 1, lane 3) was
measured via monitoring [8-14C]-guanine, [3H]-preQ1
and [3H]-queuine incorporation into E. coli tRNATyr
(fixed at 10 mM, Km = 0.19 mM, Table 4). The kcat value
for preQ1 was determined to be 9.57 ꢂ 10ꢄ3 sꢄ1, slightly
faster than that of the human enzyme (Table 2 and
Figure 4). In addition, the E. coli enzyme exhibits a low
Km (ꢃ50 nM) for preQ1, suggesting a high affinity in terms
of substrate binding. Consistent with the previous report
(33), the E. coli TGT does not seem to incorporate
queuine as a substrate as no detectable transglycosylase
activity was observed at queuine concentrations ꢅ50 mM
(data not shown). A low amount of radioactivity (not
linear with time) was captured on the filters at higher con-
centrations of queuine. However, control experiments
conducted with no substrate RNA in the reaction
mixtures confirm that the background radioactivity most
lane 2), the transglycosylase activity was measured via
monitoring [3H]-queuine and [3H]-preQ1 incorporation
into human tRNATyr. The concentration of tRNA was
fixed at saturation (10 mM), as the Km value was previ-
ously determined to be 0.34 0.04 mM (16). The kinetic
parameters were then determined by non-linear fits to the
Michaelis–Menten equation (Figure 3A and B, Table 1).
The results show that the kcat values for both substrates
are essentially identical (ꢃ8 ꢂ 10ꢄ3 sꢄ1); however,
the human TGT incorporates its natural heterocyclic
substrate, queuine, with
a
sub-micromolar Km
(0.26 0.03 mM), while a 507-fold larger Km for preQ1
(132.33 27.61 mM) was observed. Due to the indistin-
guishable kcat values, the difference in Km also leads to a
significantly higher catalytic efficiency (as defined by kcat
Km) toward queuine, clearly indicating the preferential
/
recognition of the human TGT for queuine.

2840 Nucleic Acids Research, 2011, Vol. 39, No. 7
S11A, lanes 4–6 and Figure S11B, lane 2) were generated
to probe the heterocyclic substrate recognition using [14C]-
guanine and [3H]-preQ1. The kinetic parameters for
guanine exchange were determined for both the guanine
and tRNA substrates with the alanine, serine and aspar-
tate mutants (Tables 3 and 4; also see Supplemetary
Figures S14–16 for the guanine and tRNA kinetic
curves). An increase in kcat relative to wild-type is
observed for both the Cys145Ala and Cys145Ser
mutants (14.5- and 5-fold for Cys145Ala and Cys145Ser,
respectively). The activity of the Cys145Asp mutant with
guanine is approximately equivalent to that of wild-type
TGT. Similar trends in Km were observed for both sub-
strates with the Cys145Asp and Cys145Ser mutants.
Increases, relative to wild-type, in Km of 6.7- and
5.5-fold were seen with guanine for both enzymes, and
5.8-fold with tRNA for Cys145Ala. The increase in the
tRNA Km relative to wild-type was only slightly less
(2.6-fold) with Cys145Ser. However, with the Cys145Asp
mutant, the substrate Kms are very different. While the
tRNA Km increases 17-fold, the guanine Km is 195-times
greater than that of wild-type.
Figure 4. Michaelis–Menten fit of PreQ1 with wild-type E. coli TGT.
Table 3. Guanine kinetics for wild-type and Cys145 mutant E. coli
TGTs
The kinetic parameters for preQ1 exchange were also
determined while the concentration of tRNA was main-
tained at saturating concentration (depending on the
tRNA Km for each individual mutant). Similar to
guanine kinetics, an increase in kcat was also observed
for both the Cys145Ala and Cys145Ser mutants;
however, the differences compared to the wild-type were
only 7.8- and 2.5-fold for Cys145Ala and Cys145Ser, re-
spectively (Table 2; also see Supplemetary Figures S17 and
S18 for the preQ1 kinetic plots). The enzymatic function of
Cys145Asp with respect to preQ1 appears to be abrogated
since no activity was found at preQ1 concentrations
ꢅ100 mM. Interestingly, the greatest impact on the
kinetics comes from the significant increase in Km for
preQ1 with the Cys145Ala and Cys145Ser mutants as
the values are 868- and 2460-fold higher than that of the
wild-type. The dramatic increase observed in Km indicates
that these two mutants recognize preQ1 much more poorly
than the wild-type enzyme. More intriguingly, a valine
substitution (which is the corresponding residue in
eukarya) at Cys145 only leads to a 55-fold increase in
a,b
kcat
(10ꢄ3ꢁsꢄ1
a,b
Km
(mM)
a,b
kcat/Km
(10ꢄ3ꢁsꢄ1ꢁmMꢄ1
Enzyme
)
)
Wild-type
Cys145Ala
Cys145Ser
Cys145Asp
6.29 (0.12)
86.8 (2.1)
28.0 (0.7)
5.44 (0.23)
0.35 (0.03)
2.34 (0.23)
1.94 (0.17)
68.1 (7.8)
18.0 (1.6)
37.1 (3.9)
14.4 (1.3)
0.080 (0.01)
aStandard errors are shown in parentheses.
bKinetic parameters were calculated from the average of three replicate
determinations of initial velocity data.
Table 4. tRNA kinetics for wild-type and Cys145 mutant E. coli
TGTs
a,b
kcat
(10ꢄ3ꢁsꢄ1
a,b
Km
(mM)
a,b
kcat/Km
(10ꢄ3ꢁsꢄ1ꢁmMꢄ1
Enzyme
)
)
Wild-type
Cys145Ala
Cys145Ser
Cys145Asp
5.19 (0.29)
76.3 (3.1)
28.2 (1.0)
6.34 (0.41)
0.19 (0.05)
1.11 (0.22)
0.49 (0.09)
3.30 (0.79)
27.3 (7.3)
68.7 (14.0)
58.0 (10.5)
1.92 (0.48)
Km (Table
Furthermore, a human TGT mutant, whose correspond-
ing valine (Val161) was replaced by cysteine
2
and Supplemetary Figure S19).
aStandard Errors are shown in parentheses.
bKinetic parameters were calculated from the average of three replicate
determinations of initial velocity data.
a
(SDS-PAGE in Supplemetary Figure S11B, lane 3), ex-
hibited a significant improvement in preQ1 recognition
(Figure 5) as a substantially lower Km as well as a
higher catalytic efficiency were observed.
likely resulted from either non-specific binding of queuine
to the filters (at the higher concentrations) or queuine ap-
proaching its limit of solubility, precipitating out of
solution and being captured on the filters. The kinetic par-
ameters for guanine and tRNA (Tables 3 and 4) are
similar to those previously reported (20).
DISCUSSION
It has been previously suggested that eubacterial and
eukaryal TGTs arose via convergent evolution (17) due
to real and perceived differences in the overall pathways
for queuine incorporation. More recently a divergent evo-
lution model has been proposed, which is based on careful
analyses of the X-ray crystal structures of eubacterial and
archaeal TGTs (18). The X-ray crystal structures of
Kinetics analysis of E. coli TGT Cys145 mutants
To confirm the role of active site Cys145 in the E. coli
TGT, four mutants, Cys145Ala, Cys145Ser, Cys145Asp
and Cys145Val (SDS–PAGE in Supplemetary Figure

Nucleic Acids Research, 2011, Vol. 39, No. 7 2841
Figure 5. Michaelis–Menten fit of PreQ1 with human TGT Val161Cys.
eubacterial and archaeal TGTs (8,9) reveal a common 3D
structure including absolute conservation of a structural
zinc domain (34). Sequence homologies (Figure 1) in
TGTs across the three kingdoms reveal the absolute con-
servation of the four zinc-binding ligands and three key
catalytic aspartates in the active site (Figure 6).
Eukaryotes also have a TGT-homolog (QTRTD1),
which contains the zinc-binding domain and is homolo-
gous to the TGT family, but does not share the absolute
conservation of the active-site aspartates. QTRTD1 has
been proposed to be the salvage enzyme that hydrolyzes
QMP to generate free queuine (35,36). It seems likely that
the differences in active site amino acids may be due to the
altered chemical reaction that the salvage enzyme cata-
lyzes. All TGTs across the three kingdoms are thought
to share the same chemical and kinetic mechanisms (1).
Although there is a kingdom-based difference in TGT
quaternary structure, a similar subunit structure of the
enzyme has been identified, wherein eubacterial and
archaeal TGTs form homodimers (8–10) and the
eukaryal TGT is a heterodimer of two homologous
subunits (15,16). The high degree of sequence conserva-
tion and similar dimeric subunit structures suggest that the
eukaryal TGTs will be found to share the same tertiary
structure as the eubacterial and archaeal TGTs. In this
article we sought to provide experimental evidence for
the divergent evolution of the eukaryal TGT.
A simple multiple sequence alignment of representative
TGTs across the three kingdoms (Figure 1) reflects the
absolute conservation of active-site and zinc-binding
residues as well as significant additional sequence
identity and homology. The phylogenetic tree analysis
(Figure 2) clearly demonstrates the relatedness of the
TGT sequences. The archaeal sequences grouping quite
distinctly from the others as might be expected due to
the presence of an additional domain in archaeal TGTs
versus eukaryal and eubacterial TGTs. The proximity of
the eukaryal sequences to the eubacterial sequences may
be due in part to the low number (13 versus 59 and 72) of
Figure 6. Active site of the PreQ1-bound Z. mobilis TGT (PDB acces-
sion code 1P0E). The (A) and (B) (view from two different angles) were
generated by PyMOL (The PyMOL Molecular Graphics System,
Version 1.3, Schrodinger, LLC). To be consistent with the text, the
amino acid residues were labeled based on the E. coli numbering.
Atoms oxygen, nitrogen and sulfur are highlighted in red, blue and
yellow, respectively. Note: Tyr 93 in the Z. mobilis TGT corresponds
to Phe 93 in the E. coli enzyme.
eukaryal sequences, which may not be sufficient to reveal a
larger separation between the eukaryal and eubacterial
branches. The multiple sequence alignment and phylogen-
etic tree analysis both support divergent evolution of
eubacterial and archaeal TGTs (18) and are clearly con-
sistent with the extension of this concept to the eukaryal
kingdom.
Kinetic experiments using queuine and preQ1 were per-
formed to investigate the heterocyclic substrate recogni-
tion of the human TGT. To the best of our knowledge,
this is the first report of the kinetics of the eukaryal TGT
with respect to queuine and preQ1 via direct incorporation
assays. It is surprising that both kcat and Km with respect
to queuine are not substantially different from those of
guanine [5.86 0.10 ꢂ 10ꢄ3 sꢄ1 and 0.41 0.03 mM,
(16)]. Comparison of the catalytic efficiencies (kcat/Km)
revealed that the human TGT appears to utilize queuine
only 2–3 times more efficiently than guanine (31.6
3.4 ꢂ 10ꢄ3 sꢄ1mMꢄ1 versus 14.2 0.9 ꢂ 10ꢄ3 sꢄ1mMꢄ1).
Given that queuine is the natural substrate for the
human TGT and the fact the physiological concentra-
tions are generally estimated to be in the low nanometer
range in various eukaryal tissues [e.g. ꢃ3.6 nM in
human milk (37)], a substantially lower Km and a signifi-
cantly higher kcat/Km values would have been expected.

2842 Nucleic Acids Research, 2011, Vol. 39, No. 7
However, the fact that queuine incorporation is irrevers-
ible (3) whereas guanine insertion simply regenerates the
substrate tRNA suggests that the human TGT may not
experience significant selection pressure to improve the
specificity for queuine over guanine. The slightly
improved binding affinity (or catalytic efficiency) toward
queuine is likely sufficient for organisms to efficiently
generate Q-tRNA. Moreover, since product release is
rate-limiting (5) during catalysis, a significant difference
in kcat would not be expected if the effect of queuine
(e.g., Q-tRNA versus G-tRNA product) on the tRNA
off-rate is minimal. Additionally, a recent report shows
that the two subunits of the human TGT co-localize into
mitochondria (15), suggesting that in vivo transglycosylase
activity (and hence the differential activity with guanine
versus queuine) might be affected by an association with
mitochondria which has yet to be studied.
It should be noted that the kinetic parameters reported
here for the human TGT differ (Table 1) from those pre-
viously reported by Shindo-Okada et al. (3) in 1980 for the
TGT isolated from rat liver and from those reported by
Howes and Farkas in 1978 for the TGT from rabbit
erythrocytes (11). In each of those cases, the methods
used by the authors were much less precise and quite dif-
ferent from those reported here, largely due to the state of
technology available at that time. For example, the ‘wash
out’ assay used by Shindo-Okada et al. is much less
accurate than the direct incorporation assay reported
herein as the wash out assay looks for small differences
previous work has suggested a hydrophobic interaction
between this residue and the 7-aminomethyl moiety
of the base (8,34), there has been no kinetic evidence
to support this interaction. To probe this, the alanine
mutant at this position was generated and expected
to display an attenuated hydrophobic effect due to
the smaller side chain. In contrast, serine is slightly
smaller in comparison to cysteine, but more polar
because of the large electronegativity of the oxygen
while a negatively charged aspartic acid (at physiological
pH) would likely be involved in strong electrostatic
interactions.
A few significant observations arose from the kinetic
characterization of the Cys145 mutants. First, the
parallel increases in Km values for both the guanine and
tRNA substrates (Tables 3 and 4) suggest that similar
interactions occur between this residue and guanine
whether the base is free or integrated within the tRNA
macromolecule. Second, comparing the kcat/Km values
for the enzymes with respect to preQ1 (Table 2), the
expected trend, wild-type > Cys145Val > Cys145Ala >
Cys145Ser > Cys145Asp (not actually detected) was
observed, in accordance with the crystallographic
evidence that Cys145 interacts with the base via a hydro-
phobic interaction as either polar or charged amino acid
substitutions lead to a decrease or even loss of substrate
recognition. These data are consistent with the concept
that the E. coli TGT has evolved the nature of the amino
acid at position 145 to optimize recognition of preQ1.
Moreover, the kcat/Km value for preQ1 with the human
TGT Val161Cys mutant (Table 2) is 3.4-fold higher than
that with the wild-type human enzyme. Val161 of the
human TGT corresponds to Cys145 in the E. coli TGT,
therefore this increase catalytic efficiency with respect to
preQ1 supports the concept that TGTs have divergently
evolved the amino acid at this position to specifically rec-
ognize the cognate substrate (e.g. preQ1 versus queuine).
In the case of guanine, the trend in kcat/Km is slightly
in
a
large number (e.g., observing the loss of
pre-exchanged [14C]-guanine from tRNA). In addition,
heterogenous tRNA was employed as a substrate by
Shindo-Okada et al. and only 3–4 concentrations of the
heterocyclic substrate were used to establish kinetic par-
ameters. Furthermore, those assays employed single time
points (2 h), with no assurance that the reaction was linear
out to this endpoint. Finally, a considerable amount of
error is introduced by fitting the data to a linear, double
reciprocal plot versus the non-linear fit directly to the
Michaelis–Menten equation that was used in the present
report.
different
(Cys145Ala > wild-type > Cys145Ser >
Cys145Asp) and the reason for this difference is not
obvious. The significant increase in kcat for the alanine
and serine mutants indicate that the off-rate for the
product tRNA has increased and suggests that the sub-
strate tRNA might exhibit a higher Km. This is true for the
alanine mutant, but not for the serine mutant. The serine
mutant has a kcat/Km that is very similar to that for the
wild-type. Both kcat and Km are increased by factors of 4.5
and 5.5, again consistent with faster off rates of product
tRNA and substrate guanine; however, substrate tRNA
exhibits a Km only 2.6-fold higher than that for the
wild-type TGT. It is possible that there might be a water
molecule mediating the interactions between the side chain
of Cys145Ser and guanine. In support of this hypothesis,
Klebe and co-workers have found a crystallographically
well-defined water molecule mediating a hydrogen bond
between imidazole ligands (which they extrapolate to the
N7 of G34 of the substrate tRNA) and the peptide
backbone in the active site (18).
The 510-fold lower kcat/Km for preQ1, relative to
queuine, with the human TGT indicates that the enzyme
preferentially utilizes queuine. This observation nicely
matches the homology modeling results of the eukaryal
TGT, as the two hydroxyls on the cyclopentenediol
moiety are predicted to be capable of forming hydrogen
bonds with the enzyme active site residues (34). If one
assumes that changes in Km correlate well with changes
in KD, a 507-fold increase in Km in this case would repre-
sent close to 4 kcal/mol difference in binding free energy
[as estimated by Á(ÁG) = ꢄRTÁ(lnK)]. This could rea-
sonably account for the gain in binding free energy
provided by additional hydrogen bonds. On the other
hand, the E. coli TGT utilizes preQ1 with a high affinity
(Km = 50 nM) as expected, but does not recognize queuine
at all as the cyclopentenediol moiety appears to be too
bulky to be accommodated by the enzyme (34).
To further examine heterocyclic substrate recognition, a
series of cysteine 145 mutants was generated to investigate
the role of this residue in the E. coli TGT. Although
The pKa of the aminomethyl side chain of preQ1 has
been previously determined to be 9.8 in solution (38) and
therefore, preQ1 will be protonated at physiological pH,

Nucleic Acids Research, 2011, Vol. 39, No. 7 2843
enabling it to participate in a possible electrostatic inter-
action with active site residues (Figure 6). However, the
severely reduced recognition of preQ1 by the aspartate
mutant clearly rejects an electrostatic interaction with
the residue at 145. Crystallographic evidence has shown
that the amino group of the preQ1 side chain forms a
hydrogen bond with the carboxyl oxygen of Leu215
(E. coli numbering) (34). A hydrogen bond with the
protonated preQ1 side chain would provide a substantial
SUPPLEMENTARY DATA
Supplementary data are available at NAR online.
ACKNOWLEDGEMENTS
The authors thank Stefanie V. Stachura and Dr. Julie K.
Hurt for preparing the constructs of polyhistidine-tagged
E. coli TGT mutants. They also wish to acknowledge
Moravek Biochemicals for conducting the tritiation for
the [3H]-preQ1 and [3H]-queuine syntheses.
contribution (greater than
a
simple, non-charged
hydrogen bond) to the binding free energy. Another inter-
esting fact is that both the serine and alanine mutants
exhibited an increase in kcat. The TGT-catalyzed
FUNDING
reaction is extremely slow (approximately one turnover
every 2 minutes). Thus, the increases in activity exhibited
by those two mutants are significant. However, this obser-
vation is not surprising considering product release is
rate-limiting (5). Since Cys145 directly interacts with the
base, removal of this interaction would enable faster
product release. In sum, these observations are consistent
with the postulate that Cys145 evolved in eubacteria to
selectively recognize preQ1.
University of Michigan, College of Pharmacy, Vahlteich
and UpJohn Research funds (to G.A.G. and to
H.D.H.S.). The open access publication charge for this
paper has been partially waived by Oxford University
Press - NAR Editorial Board members are entitled to
one free paper per year in recognition of their work on
behalf of the journal.
Conflict of interest statement. None declared.
The fact that the eubacterial and eukaryal TGTs select-
ively recognize their cognate heterocyclic substrates is
evolutionarily significant. Unlike eubacteria, which
biosynthesize queuine from its precursor preQ1, eukarya
lack a queuine biosynthesis pathway and are required to
obtain queuine from diet as a nutrient factor (39) or
through a salvage mechanism that is yet to be fully
characterized (40). In addition, while absent in eubacteria,
a specific transport system responsible for the cellular
uptake of queuine was observed in human fibroblasts
and the process appears to be modulated by protein
kinase C (PKC) phosphorylation (41,42). We conclude
that these differences are most likely due to an out-
come of evolution, while the origin of eukarya somehow
lost the ability to produce Q-tRNA from preQ1-tRNA.
Since the incorporation of preQ1 would not lead to
the generation of Q-tRNA, it is likely that due to selec-
tion pressure, the eukaryal TGT evolved to take
up queuine directly in order to compensate for the in-
ability to synthesize queuine de novo. It is also an interest-
ing note that there is a 6-fold difference in kcat /Km
between the human and E. coli TGTs with respect to
their cognate substrates (Tables 1 and 2). Considering
eubacteria need to complete the queuosine biosynthesis
pathway to obtain Q-tRNA while eukarya do not, the
greater efficiency of the eubacterial TGT seems perfectly
plausible.
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