Mechanistic Studies of the tRNA-Modifying Enzyme QueA:  A Chemical Imperative for the Use of AdoMet as a “Ribosyl” Donor

Kinzie, Sylvia Daoud; Thern, Bernd; Iwata‐Reuyl, Dirk

doi:10.1021/ol005756h

Cited by 38 publications

(23 citation statements)

References 32 publications

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“…The reaction catalyzed by QueA is unprecedented in biological systems, and the results of preliminary mechanistic studies are consistent with a catalytic mechanism involving sulfonium ylide and vinyl sulfonium intermediates (39), species never before implicated in an enzymatic reaction. However, in contrast to TGT, the lack of an easily accessible activity assay has stymied the basic characterization and quantitative analysis of this remarkable enzyme and precluded detailed kinetic studies.…”

supporting

confidence: 66%

tRNA Modification by S-Adenosylmethionine:tRNA Ribosyltransferase-Isomerase

Lanen

Kinzie

Matthieu

et al. 2003

Journal of Biological Chemistry

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The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase catalyzes the penultimate step in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q), an unprecedented ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor to generate epoxyqueuosine (oQ). The complexity of the reaction makes it an especially interesting mechanistic problem, and as a foundation for detailed kinetic and mechanistic studies we have carried out the basic characterization of the enzyme. Importantly, to allow for the direct measurement of oQ formation, we have developed protocols for the preparation of homogeneous substrates; specifically, an overexpression system was constructed for tRNA Tyr in an E. coli queA deletion mutant to allow for the isolation of large quantities of substrate tRNA, and [U-ribosyl-14 C]AdoMet was synthesized. The enzyme shows optimal activity at pH 8.7 in buffers containing various oxyanions, including acetate, carbonate, EDTA, and phosphate. Unexpectedly, the enzyme was inhibited by Mg 2؉and Mn 2؉ in millimolar concentrations. The steady-state kinetic parameters were determined to be K m AdoMet ‫؍‬ 101.4 M, K m tRNA ‫؍‬ 1.5 M, and k cat ‫؍‬ 2.5 min ؊1 . A short minihelix RNA was synthesized and modified with the precursor 7-aminomethyl-7-deazaguanine, and this served as an efficient substrate for the enzyme (K m RNA ‫؍‬ 37.7 M and k cat ‫؍‬ 14.7 min ؊1 ), demonstrating that the anticodon stem-loop is sufficient for recognition and catalysis by QueA.

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confidence: 66%

tRNA Modification by S-Adenosylmethionine:tRNA Ribosyltransferase-Isomerase

Lanen

Kinzie

Matthieu

et al. 2003

Journal of Biological Chemistry

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“…Pre-Q 1 is subsequently inserted into tRNA by the enzyme tRNAguanine transglycosylase (TGT; EC 2.4.2.29) (36,37), which is encoded in E. coli by the tgt gene (35). The rest of the biosynthesis of Q occurs at the level of the tRNA and involves the formation of epoxyqueuosine by S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA; EC 2.4.2.-) (22,45,46), followed by reduction of the epoxide in epoxyqueuosine by an as-yet-unidentified enzyme to give Q (13).…”

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confidence: 99%

Biosynthesis of 7-Deazaguanosine-Modified tRNA Nucleosides: a New Role for GTP Cyclohydrolase I

et al. 2008

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Queuosine (Q) and archaeosine (G ؉ ) are hypermodified ribonucleosides found in tRNA. Q is present in the anticodon region of tRNA GUN in Eukarya and Bacteria, while G ؉ is found at position 15 in the D-loop of archaeal tRNA. Prokaryotes produce these 7-deazaguanosine derivatives de novo from GTP through the 7-cyano-7-deazaguanine (pre-Q 0 ) intermediate, but mammals import the free base, queuine, obtained from the diet or the intestinal flora. By combining the results of comparative genomic analysis with those of genetic studies, we show that the first enzyme of the folate pathway, GTP cyclohydrolase I (GCYH-I), encoded in Escherichia coli by folE, is also the first enzyme of pre-Q 0 biosynthesis in both prokaryotic kingdoms. Indeed, tRNA extracted from an E. coli ⌬folE strain is devoid of Q and the deficiency is complemented by expressing GCYH-I-encoding genes from different bacterial or archaeal origins. In a similar fashion, tRNA extracted from a Haloferax volcanii strain carrying a deletion of the GCYH-I-encoding gene contains only traces of G ؉ . These results link the production of a tRNA-modified base to primary metabolism and further clarify the biosynthetic pathway for these complex modified nucleosides.It is well established that GTP is not only a molecule central to energy conservation and a precursor to the synthesis of RNA and DNA but also the precursor to a number of essential metabolites. These include riboflavin and the pterin-related coenzymes tetrahydropterin (BH4), tetrahydrofolate (THF), methanopterin, and molybdopterin. It is less well known that GTP is also the precursor of the 7-deazapurine derivatives found in tRNA (queuosine [Q] and archaeosine

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“…Introduction of queuosine into tRNAs is initiated when a transglycosylase, encoded by the tgt gene, exchanges the guanine base at the first position of the GUN anticodon in tRNAs with preQ 1 , a precursor of the base (queuine) and queuosine. QueA then attaches a substituted cyclopentenyl group to preQ 1 to produce epoxyqueuosine, which is followed by a B 12 -dependent step in which the epoxide in epoxyqueuosine is reduced, yielding queuosine (20,24,25,33,36,37).…”

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Environmental and Growth Phase Regulation of the Streptococcus gordonii Arginine Deiminase Genes

Liu

Dong

Chen

et al. 2008

Appl Environ Microbiol

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A 1,026-bp open reading frame sharing significant similarity with queA, which encodes a predicted Sadenosylmethionine:tRNA ribosyltransferase-isomerase responsible for queosine modification of tRNAs, was found immediately 5 of the gene for the transcriptional activator (ArcR) of the arginine deiminase system (ADS) operon of Streptococcus gordonii. The role of QueA in bacterial physiology is enigmatic, but loss of QueA has been shown to compromise stationary-phase survival or virulence in certain enteric bacteria. Interestingly, S. gordonii appears to be unique among ADS-positive bacteria in the linkage of queA with the ADS genes. A putative 70 promoter (p queA ; TTGCCA-N 21 -TATAAT) was mapped 5 of queA by primer extension, and queA and arcR were shown to be cotranscribed. The expression from p queA was found to be constitutive under all conditions tested, but the expression of p arcA , which drives the expression of the arc structural genes, was enhanced in stationary phase and could be induced by low pH and arginine. QueA and CcpA acted repressively on arc transcription, but neither QueA-deficient strains nor CcpA-deficient strains showed significant differences in arginine deiminase enzyme activities compared with the wild-type strain. The growth rate of a QueA-deficient strain did not differ significantly from that of the parental strain, but the QueA-deficient strain did not compete well with the wild-type during serial passage. In addition to the finding that ADS expression can be regulated separately by growth phase and pH, a significant linkage between the ADS, translational efficiency modulated by QueA, and post-exponential-phase survival of S. gordonii was found.

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Mechanistic Studies of the tRNA-Modifying Enzyme QueA: A Chemical Imperative for the Use of AdoMet as a “Ribosyl” Donor

Cited by 38 publications

References 32 publications

tRNA Modification by S-Adenosylmethionine:tRNA Ribosyltransferase-Isomerase

tRNA Modification by S-Adenosylmethionine:tRNA Ribosyltransferase-Isomerase

Biosynthesis of 7-Deazaguanosine-Modified tRNA Nucleosides: a New Role for GTP Cyclohydrolase I

Environmental and Growth Phase Regulation of the Streptococcus gordonii Arginine Deiminase Genes

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