Publications

• Transfer and isomerization of the ribose moiety of AdoMet during the biosynthesis of queuosine tRNAs, a new unique reaction catalyzed by the QueA protein from Escherichia coli.

  Slany R.K., Boesl M., Kersten H.

  The enzyme QueA of E coli is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine. The enzyme catalyzes the synthesis of an epoxycyclopentane moiety and transfers this compound to specific tRNAs containing the queuosine precursor 7-(aminomethyl)-7-deazaguanine (preQ1). S-ade ... >> More

  The enzyme QueA of E coli is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine. The enzyme catalyzes the synthesis of an epoxycyclopentane moiety and transfers this compound to specific tRNAs containing the queuosine precursor 7-(aminomethyl)-7-deazaguanine (preQ1). S-adenosylmethionine (AdoMet) is the sole cofactor that is required for this reaction (Slany et al, 1993, Biochemistry 32, 7811-7817). To proof that the ribose moiety of AdoMet is the precursor of the epoxycyclopentane moiety, labeled AdoMet, was generated from different types of 3H ATP and methionine by the AdoMet synthetase enzyme (MetK) from E coli. The resulting 3H labeled AdoMet was directly used as the cofactor for the QueA reaction. Using [2,5', 8-3H]ATP, containing tritium at C5' of the ribose ring, resulted in an incorporation of radioactivity into preQ1 tRNA, whereas this was not the case when [2,8-3H]ATP was applied. A model for the reaction catalyzed by the S-adenosylmethionine:tRNA ribosyltransferase-isomerase QueA is proposed. << Less

  Biochimie 76:389-393(1994) [PubMed] [EuropePMC]

• Mechanistic studies of the tRNA-modifying enzyme QueA: a chemical imperative for the use of AdoMet as a "ribosyl" donor.

  Kinzie S.D., Thern B., Iwata-Reuyl D.

  [formula: see text] The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) catalyzes the penultimate step in the biosynthesis of the tRNA nucleoside queuosine, a unique ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor. The use of AdoMe ... >> More

  [formula: see text] The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) catalyzes the penultimate step in the biosynthesis of the tRNA nucleoside queuosine, a unique ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor. The use of AdoMet in this way is fundamentally new to the chemistry of this important biological cofactor. We report here the first mechanistic studies of this remarkable enzyme, and we propose a chemical mechanism for the reaction consistent with our experimental observations. << Less

  Org Lett 2:1307-1310(2000) [PubMed] [EuropePMC]

• Kinetic mechanism of the tRNA-modifying enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA).

  Van Lanen S.G., Iwata-Reuyl D.

  The bacterial enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) catalyzes the unprecedented transfer and isomerization of the ribosyl moiety of S-adenosylmethionine (AdoMet) to a modified tRNA nucleoside in the biosynthesis of the hypermodified nucleoside queuosine. The complexi ... >> More

  The bacterial enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) catalyzes the unprecedented transfer and isomerization of the ribosyl moiety of S-adenosylmethionine (AdoMet) to a modified tRNA nucleoside in the biosynthesis of the hypermodified nucleoside queuosine. The complexity of this reaction makes it a compelling problem in fundamental mechanistic enzymology, and as part of our mechanistic studies of the QueA-catalyzed reaction, we report here the elucidation of the steady-state kinetic mechanism. Bi-substrate kinetic analysis gave initial velocity patterns indicating a sequential mechanism, and provided the following kinetic constants: K (M)(tRNA)= 1.9 +/-0.7 microM and K (M)(AdoMet)= 98 +/-5.0 microM. Dead-end inhibition studies with the substrate analogues S-adenosylhomocysteine and sinefungin gave competitive inhibition patterns against AdoMet and noncompetitive patterns against preQ(1)-tRNA(Tyr), with K(i) values of 133 +/- 18 and 4.6 +/-0.5 microM for sinefungin and S-adenosylhomocysteine, respectively. Product inhibition by adenine was noncompetitive against both substrates under conditions with a subsaturating cosubstrate concentration and uncompetitive against preQ(1)-tRNA(Tyr) when AdoMet was saturating. Inhibition by the tRNA product (oQ-tRNA(Tyr)) was competitive and noncompetitive against the substrates preQ(1)-tRNA(Tyr) and AdoMet, respectively. Inhibition by methionine was uncompetitive versus preQ(1)-tRNA(Tyr), but noncompetitive against AdoMet. However, when methionine inhibition was investigated at high AdoMet concentrations, the pattern was uncompetitive. Taken together, the data are consistent with a fully ordered sequential bi-ter kinetic mechanism in which preQ(1)-tRNA(Tyr) binds first followed by AdoMet, with product release in the order adenine, methionine, and oQ-tRNA. The chemical mechanism that we previously proposed for the QueA-catalyzed reaction [Daoud Kinzie, S., Thern, B., and Iwata-Reuyl, D. (2000) Org. Lett. 2, 1307-1310] is consistent with the constraints imposed by the kinetic mechanism determined here, and we suggest that the magnitude of the inhibition constants for the dead-end inhibitors may provide insight into the catalytic strategy employed by the enzyme. << Less

  Biochemistry 42:5312-5320(2003) [PubMed] [EuropePMC]

• A new function of S-adenosylmethionine: the ribosyl moiety of AdoMet is the precursor of the cyclopentenediol moiety of the tRNA wobble base queuine.

  Slany R.K., Boesl M., Crain P.F., Kersten H.

  Queuosine (Q) [7-(((4,5-cis-dihydroxy-2-cyclopenten-1-yl)amino)methyl)-7-deaz agu anosine] usually occurs in the first position of the anticodon of tRNAs specifying the amino acids asparagine, aspartate, histidine, and tyrosine. The hypermodified nucleoside is found in eubacteria and eucaryotes. Q ... >> More

  Queuosine (Q) [7-(((4,5-cis-dihydroxy-2-cyclopenten-1-yl)amino)methyl)-7-deaz agu anosine] usually occurs in the first position of the anticodon of tRNAs specifying the amino acids asparagine, aspartate, histidine, and tyrosine. The hypermodified nucleoside is found in eubacteria and eucaryotes. Q is synthesized de novo exclusively in eubacteria; for eucaryotes the compound is a nutrient factor. In Escherichia coli the Q precursor (oQ), carrying a 2,3-epoxy-4,5-dihydroxycyclopentane ring, is formed from tRNA precursors containing 7-(aminomethyl)-7-deazaguanine (preQ1) by the queA gene product. A genomic queA mutant accumulating preQ1 tRNA was constructed. The QueA enzyme was overexpressed as a fusion protein with the glutathione S-transferase from Schistosoma japonicum and purified to homogeneity by affinity and anion-exchange chromatography. The enzyme QueA synthesizes oQ from preQ1 in a single S-adenosylmethionine-(AdoMet-) requiring step, indicating that the ribosyl moiety of AdoMet is transferred and isomerized to the epoxycyclopentane residue of oQ. The identity of oQ was verified by HPLC and directly combined HPLC/mass spectrometry. The formation of oQ was reconstituted in vitro, applying a synthetic RNA. A 17-nucleotide microhelix (corresponding to the anticodon stem and loop of tRNA(Tyr) from E. coli) is sufficient to act as the RNA substrate for oQ synthesis. We propose that QueA is an S-adenosylmethionine:tRNA ribosyltransferase-isomerase. << Less

  Biochemistry 32:7811-7817(1993) [PubMed] [EuropePMC]