Decoding the logic of the tRNA regiospecificity of nonribosomal femX Wv aminoacyl transferase www.angewandte.org

Article abstract of DOI:10.1002/anie.201001473

Natural selection: Replacement of the 3′-OH group of Ala-tRNA ^Ala with 3′-H affected FemX_Wv-catalyzed aminoacyl transfer from the 2 -position, but not substrate binding. The ability of FemX_Wv to bind and transacylate the 3′-O-Ala isomer initially formed by alanyl-tRNA synthetase (AlaRS) may be crucial for efficient competition with the ribosome (see scheme). (Figure Presented).

Full text of DOI:10.1002/anie.201001473

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Chemie
DOI: 10.1002/anie.201001473
Aminoacyl-tRNA Mimics
Decoding the Logic of the tRNA Regiospecificity of Nonribosomal
FemXWv Aminoacyl Transferase**
Matthieu Fonvielle, Maryline Chemama, Maxime Lecerf, Rꢀgis Villet, Patricia Busca,
Ahmed Bouhss, Mꢀlanie Ethꢁve-Quelquejeu,* and Michel Arthur*
Aminoacyl-tRNAs are key intermediates in protein synthesis.
They act as adapters between the codons of mRNA and the
growing polypeptide chain in the ribosome.[1] The vicinal
hydroxy groups at the 2’- and 3’-positions of the terminal
nucleotide (A76) of tRNA have pivotal roles in the function of
these molecules. The tRNA molecules are esterified by
aminoacyl-tRNA synthetases,[2] which catalyze the transfer
of a specific aminoacyl residue from an adenylate to the 2’- or
3’-hydroxy group of A76 (Scheme 1). Transesterification
between the 2’- and 3’-positions occurs in the absence of an
enzyme with a rate and thermodynamic equilibrium of the
order of 5 sÀ1 and 1, respectively.[3] The A site of the ribosome
is specific for the 3’-O-aminoacyl isomer, and the 3’ linkage to
the tRNA is conserved in the product of the peptidyl-transfer
reaction.[4] The 2’-hydroxy group of the peptidyl-tRNA is
thought to assist catalysis of this reaction.[5]
Besides their role in protein synthesis, aminoacyl-tRNAs
participate in various metabolic pathways,[6] such as the
synthesis of cyclodipeptides[7] or the aminoacylation of
proteins[8] and membrane phosphatidylglycerol.[9] Transfer-
ases of the Fem family catalyze the incorporation of amino
acids into peptidoglycan precursors to form a side chain that
contains the amino group used as an acyl acceptor in the final
cross-linking step of cell-wall synthesis[10] (Scheme 1). The
specificity of these enzymes is essential for bacteria, since
misincorporated amino acids can act as chain terminators and
block peptidoglycan polymerization.[10] Because of their key
role in peptidoglycan metabolism, Fem transferases are
considered attractive targets for the development of novel
antibiotics.[10]
We previously used chemical acylation[11] of RNA helices
with natural and nonproteinogenic amino acids to gain insight
into the specificity of FemXWv of Weissella viridescens,[12,13]
a
model enzyme of the Fem family.[14] A combination of
modifications in the RNA and aminoacyl moieties of the
substrate revealed that unfavorable interactions of FemXWv
with the acceptor arm of tRNAGly and with l-Ser or larger
residues quantitatively account for the preferential transfer of
l-Ala observed with complete aminoacyl-tRNAs.[12,13] The
main FemXWv identity determinant of Ala-tRNAAla was
found to be the penultimate base pair, G2–C71, which is
replaced with C2–G71 in tRNAGly isoacceptors.[12,13]
In this study, we synthesized nonisomerizable mimics of
Ala-tRNAAla that contained 2’-deoxyadenosine or 3’-deoxy-
adenosine to lock the amino acid in the 3’- and 2’-position,
respectively (Scheme 2). We also synthesized nonisomeriz-
able aminoacyl-tRNA analogues by replacing the ester bond
connecting the amino acid residue to the terminal nucleotide
with a triazole ring[15] (Scheme 3). We synthesized these
molecules to determine the regiospecificity of FemXWv for the
3’ and 2’ isomers and to evaluate the role of the adjacent
hydroxy group in the transfer reaction.
[*] Dr. M. Fonvielle,[+] M. Lecerf, Dr. R. Villet, Dr. M. Arthur
Laboratoire de Recherche Molꢀculaire sur les Antibiotiques
Centre de Recherche des Cordeliers, Equipe 12, UMR S 872
INSERM, Universitꢀ Pierre et Marie Curie—Paris 6 and
Universitꢀ Paris Descartes
Ala-tRNAAla analogues containing a terminal 2’- or 3’-
deoxyadenosine residue and a 24 nucleotide (nt) helix
mimicking the acceptor arm of the tRNA (Figure 1) were
obtained by semisynthesis (see the Supporting Information
and Scheme 2) and assayed as substrates of FemXWv
15, rue de l’Ecole de Mꢀdecine, 75006 Paris (France)
Fax: (+33)1-4325-6812
E-mail: michel.arthur@crc.jussieu.fr
Dr. M. Chemama,[+] Dr. P. Busca, Dr. M. Ethꢁve-Quelquejeu
Institut Parisien de Chimie Molꢀculaire, CNRS UMR 7201
Universitꢀ Pierre et Marie Curie—Paris 6
4, place Jussieu, 75005 Paris (France)
Fax: (+33)1-4427-5504
E-mail: melanie.etheve@upmc.fr
Dr. A. Bouhss
Laboratoire des Enveloppes Bactꢀriennes et Antibiotiques
Institut de Biochimie et de Biophysique Molꢀculaire et Cellulaire
UMR 8619, CNRS, Universitꢀ Paris-Sud, 91405 Orsay (France)
[+] These authors contributed equally to this work.
[**] This research was supported by the European Community (EUR-
INTAFAR, Project No. LSHM-CT-2004-512138, 6th PCRD) and by
the Fondation Recherche Mꢀdicale (“fin de thꢁse” to R.V. and M.C.).
FemXWv is the FemX alanyl transferase of Weissella viridescens.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201001473.
Figure 1. Analogues of Ala-tRNAAla contained a 24 nt helix to mimic
the acceptor arm of the tRNA (helixAla).
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used inefficiently as a substrate by FemXWv
.
Inhibition of the transferase activity of
FemXWv by B indicates that the presence
of Ala at the 3’-position did not prevent
binding of the Ala-tRNAAla analogue to the
enzyme, although aminoacyl transfer was
not catalyzed from this position. Thus,
FemXWv can recruit the 3’-O-aminoacyl
regioisomer produced by AlaRS. Transacy-
lation within the active site is predicted to
provide efficient access to the 2’-O-amino-
acyl regioisomer required for Ala transfer
to the peptidoglycan precursor. FemXWv
-
catalyzed transacylation is also predicted to
prevent inhibition by the 3’-O-aminoacyl
regioisomer.
To investigate the inhibition of FemXWv
by the product of the reaction, the deacy-
lated tRNAAla, we exploited the capacity of
the enzyme to transfer seryl residues from
Ser-tRNASer (see Figure 2f in the Support-
ing Information). Compound D, the unacy-
lated helixAla, inhibited this reaction mod-
erately, with an apparent IC50 value of (89 Æ
4) mm (Figure 3): a 360-fold difference in
comparison to inhibition by regioisomer A,
which contained a 2’-O-Ala ester [(0.25 Æ
0.04) mm;
Figure 2].
Compound
E
(Scheme 2), which contained a terminal 2’-
deoxyadenosine residue, inhibited FemXWv
with an IC50 value of (5.5 Æ 0.5) mm. Thus, a
free 2’-hydroxy group in the terminal
nucleotide was critical for release of the
tRNAAla product following catalysis. This
feature is also expected to contribute to the
in vivo efficiency of the enzyme by prevent-
Scheme 1. Participation of Ala-tRNAAla regioisomers in protein and peptidoglycan synthesis.
Highlighted zones show reactions catalyzed by the enzymes indicated in blue. FemXWv
transfers an alanyl residue from Ala-tRNAAla to the side chain of l-Lys in the peptidoglycan
precursor UDP-N-acetylmuramyl pentapeptide. This reaction initiates synthesis of the l-Ala-
l-Ser-l-Ala side chain, which is completed by other Fem transferases.[10] R stands for glycan
chains composed of alternating b(1,4)-N-acetylglucosamine and N-acetylmuramic acid.
UDP=uridine diphosphate.
ing inhibition by unacylated tRNAAla
To further analyze the regiospecificity of
.
FemXWv
,
we synthesized two stable
regioisomers of Ala-tRNAAla containing a
triazole ring to mimic the ester linkage
(Scheme 3). These analogues, F and G,
(Figure 2). Ala was transferred from the 2’-position of
compound A with a turnover number of (0.19 Æ 0.03) minÀ1.
FemXWv catalyzed aminoacyl transfer only from this position,
since compound B was not a substrate. The 3’-hydroxy group
was not essential for catalysis, although a 240-fold decrease in
the turnover number was observed in comparison with C,
which contained a terminal adenosine residue. Since alanyl-
tRNA synthetase (AlaRS) aminoacylates the 3’-position of
tRNAAla, transacylation of Ala must occur prior to transfer to
the peptidoglycan precursor.
which retained a vicinal hydroxy group but could not undergo
regioisomerization, inhibited FemXWv with similar IC50 values
[(2.3 Æ 0.1) and (2.4 Æ 0.4) mm, respectively; Figure 3]. This
result was expected, since regioisomers A and B inhibited
FemXWv (Figure 2). Together, these results indicate that the
240-fold decrease observed upon removal of the 3’-hydroxy
group from the substrate (compare A and C in Figure 2)
affects the enzyme-catalyzed chemical reaction rather than
substrate binding. The 3’-hydroxy group is therefore likely to
play a role in substrate-assisted catalysis. A model proposing
its participation in proton shuttling during catalysis is
presented in Scheme 1. Asp108 of FemXWv has been proposed
to act as a general base;[14] however, this residue is not located
within the enzyme active site.[16] Substrate-assisted catalysis
might account for the inability to identify any catalytic
residue.[16]
The Ala-helixAla derivatives containing a terminal 3’- or 2’-
deoxyadenosine residue were also tested as inhibitors of
FemXWv in the presence of the complete Ala-tRNAAla (76 nt)
as the substrate (Figure 2). Compound B (2’-deoxy), which
was not a substrate of FemXWv, inhibited the reaction with an
IC50 value of (0.80 Æ 0.03) mm. A threefold lower IC50 value
[(0.25 Æ 0.04) mm] was observed for A (3’-deoxy), which was
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Scheme 2. a) 1. a-acetoxyisobutyryl bromide, then Dowex OHÀ, 1 h;
2. TBSCl, pyridine, 3 h, RT; 3. LiEt3BH, THF, RT, 3 h; 4. BzCl, pyridine,
RT, 3 h; 5. TBAF, THF, AcOH, RT, 3 h; b) 1. Ac-dC-CE phosphorami-
dite, tetrazole, CH2Cl2, RT, 1 h; 2. I2, RT, 30 min; 3. TCA, CH2Cl2, RT,
30 min; 4. bis(2-cyanoethyl)diisopropylphosphoramidite, tetrazole,
CH2Cl2, RT, 1 h; 5. I2, RT, 30 min; 6. CH3NH2, RT, 24 h; 7. Dowex ion-
exchange beads in the tetrabutylammonium form; c) N-(4-pentenoyl)-
S-alanine cyanomethyl ester, DMF, CH3CN, RT, 24 h; d) 1. 22 nt RNA
helixAla, T4 RNA ligase; 2. I2, THF/H2O; e) 1. TBSCl, pyridine, RT, 4 h;
2. BzCl, pyridine, RT, 8 h; 3. TBAF, THF, AcOH, RT, 4 h. Ac-dC-CE
phosphoramidite=5’-dimethoxytrityl-N-acetyl-2’-deoxycytidine-3’-(2-
cyanoethyl-N,N-diisopropyl)phosphoramidite, Bz=benzoyl,
DMF=N,N-dimethylformamide, pdC=5’-monophosphate-2’-deoxycy-
tidine, TBAF=tetrabutylammonium fluoride, TBS=tert-butyldimethyl-
silyl, TCA=trichloroacetic acid.
Scheme 3. a) NaBH4, EtOH/H2O, 30 min; b) TfCl, DMAP, CH2Cl2, 1 h; c) NaN3, DMF, 48 h; d) TFA, H2O/THF, 08C, 24 h; e) tert-butylbut-3-yn-2-
ylcarbamate, CuSO4, sodium ascorbate, THF, H2O, RT, 24 h; f) 1. Ac-dC-CE phosphoramidite, tetrazole, CH2Cl2, RT, 1 h; 2. I2, 30 min, RT; 3. TCA,
CH2Cl2, RT, 30 min; 4. bis(2-cyanoethyl)diisopropylphosphoramidite, tetrazole, CH2Cl2, RT, 1 h; 5. I2, RT, 30 min; 6. CH3NH2, RT, 24 h; 7. HCl
(6n)/THF/CH3OH, 24 h; g) 1. 22 nt RNA helixAla, T4 RNA ligase; 2. I2, THF/H2O. DMAP=4-dimethylaminopyridine, Tf=trifluoromethanesulfonyl,
TFA=trifluoroacetic acid.
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Figure 3. Inhibition of FemXWv by helixAla containing unacylated adeno-
sine (compound D), 2’-deoxyadenosine (compound E), or a 1,2,3-
triazole ring linked to the 2’- (compound F) or 3’-position (compound
G) of adenosine.
peptidoglycan synthesis. Efficient binding of the two
regioisomers to FemXWv implies that transacylation occurs
within the active site to prevent inhibition by the 3’-O-
aminoacyl regioisomer. We previously established that the
specificity of FemXWv for Ala-tRNAAla does not depend upon
an interaction with the methyl side chain of Ala.[13] In
agreement with this conclusion, we have shown herein that
FemXWv is inhibited to a similar extent by the two Ala-
tRNAAla regioisomers. In spite of the absence of an inter-
action between the enzyme and the side chain of Ala, FemXWv
is not strongly inhibited by the deacylated product of the
aminoacyl-transfer reaction. The free 2’-hydroxy group gen-
erated by this reaction appears to be critical both in
promoting the release of the product of the reaction and in
preventing inhibition by unacylated tRNA molecules present
in the cytoplasm.
Figure 2. Analogues of Ala-tRNAAla containing a terminal 3’-deoxyade-
nosine (compound A), 2’-deoxyadenosine (compound B), or adenosine
residue (compound C) were assayed as substrates (top left graph) and
inhibitors (top right graph) of FemXWv. The magnification of different
ranges of FemXWv concentrations (bottom) shows the linear relation-
ship between the concentration of FemXWv and the concentration of
the UDP-MurNAc hexapeptide produced by the aminoacyl-transfer
reaction. These ranges were used to calculate the turnover numbers
for compounds A and C. Compound B was not a substrate. Compound
C could not be assayed as an inhibitor, since it was used efficiently by
FemXWv as a substrate. N.A.=not applicable.
The 2’-hydroxy group of peptidyl-tRNA at the P site of
the ribosome was reported to be essential, since the use of a
2’-deoxy analogue resulted in a 106-fold decrease in the rate of
peptide-bond formation.[5,17] The critical role of the 2’-
hydroxy group was proposed to reflect substrate-assisted
catalysis in the peptidyltransferase center of the ribosome.[5,17]
According to this proposal, the ribosome provides a template
for entropic activation rather than groups for acid–base
catalysis.[18] However, this model remains controversial, since
engineered suppressor tRNAs containing a 2’-deoxyadeno-
sine[19] or diacylated adenosine[20] residue were used effi-
ciently in in vitro transcription–translation coupled systems.
In conclusion, analysis of the tRNA regiospecificity of
FemXWv revealed several features that are relevant to the
physiological role of the enzyme. FemXWv and the ribosome
compete for the same pool of aminoacyl-tRNAs (Scheme 1).
Received: March 11, 2010
Published online: June 22, 2010
Keywords: enzyme catalysis · peptides · transesterification ·
.
transferases · tRNA mimics
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