Publications

• Identification of eukaryotic and prokaryotic methylthiotransferase for biosynthesis of 2-methylthio-N6-threonylcarbamoyladenosine in tRNA.

  Arragain S., Handelman S.K., Forouhar F., Wei F.Y., Tomizawa K., Hunt J.F., Douki T., Fontecave M., Mulliez E., Atta M.

  Bacterial and eukaryotic transfer RNAs have been shown to contain hypermodified adenosine, 2-methylthio-N(6)-threonylcarbamoyladenosine, at position 37 (A(37)) adjacent to the 3'-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. Using a ... >> More

  Bacterial and eukaryotic transfer RNAs have been shown to contain hypermodified adenosine, 2-methylthio-N(6)-threonylcarbamoyladenosine, at position 37 (A(37)) adjacent to the 3'-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. Using a combination of bioinformatic sequence analysis and in vivo assay coupled to HPLC/MS technique, we have identified, from distinct sequence signatures, two methylthiotransferase (MTTase) subfamilies, designated as MtaB in bacterial cells and e-MtaB in eukaryotic and archaeal cells. Both subfamilies are responsible for the transformation of N(6)-threonylcarbamoyladenosine into 2-methylthio-N(6)-threonylcarbamoyladenosine. Recently, a variant within the human CDKAL1 gene belonging to the e-MtaB subfamily was shown to predispose for type 2 diabetes. CDKAL1 is thus the first eukaryotic MTTase identified so far. Using purified preparations of Bacillus subtilis MtaB (YqeV), a CDKAL1 bacterial homolog, we demonstrate that YqeV/CDKAL1 enzymes, as the previously studied MTTases MiaB and RimO, contain two [4Fe-4S] clusters. This work lays the foundation for elucidating the function of CDKAL1. << Less

  J. Biol. Chem. 285:28425-28433(2010) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Mechanism of N6-threonylcarbamoyladenosine (t(6)A) biosynthesis: isolation and characterization of the intermediate threonylcarbamoyl-AMP.

  Lauhon C.T.

  Genetic and biochemical studies have recently implicated four proteins required in bacteria for the biosynthesis of the universal tRNA modified base N6-threonylcarbamoyl adenosine (t(6)A). In this work, t(6)A biosynthesis in Bacillus subtilis has been reconstituted in vitro and found to indeed req ... >> More

  Genetic and biochemical studies have recently implicated four proteins required in bacteria for the biosynthesis of the universal tRNA modified base N6-threonylcarbamoyl adenosine (t(6)A). In this work, t(6)A biosynthesis in Bacillus subtilis has been reconstituted in vitro and found to indeed require the four proteins YwlC (TsaC), YdiB (TsaE), YdiC (TsaB) and YdiE (TsaD). YwlC was found to catalyze the conversion of L-threonine, bicarbonate/CO(2) and ATP to give the intermediate L-threonylcarbamoyl-AMP (TC-AMP) and pyrophosphate as products. TC-AMP was isolated by HPLC and characterized by mass spectrometry and (1)H NMR. NMR analysis showed that TC-AMP decomposes to give AMP and a nearly equimolar mixture of L-threonine and 5-methyl-2-oxazolidinone-4-carboxylate as final products. Under physiological conditions (pH 7.5, 37 °C, 2 mM MgCl(2)), the half-life of TC-AMP was measured to be 3.5 min. Both YwlC (in the presence of pyrophosphatase) and its Escherichia coli homologue YrdC catalyze the formation of TC-AMP while producing only a small molar fraction of AMP. This suggests that CO(2) and not an activated form of bicarbonate is the true substrate for these enzymes. In the presence of pyrophosphate, both enzymes catalyze clean conversion of TC-AMP back to ATP. Purified TC-AMP is efficiently processed to t(6)A by the YdiBCE proteins in the presence of tRNA substrates. This reaction is ATP independent in vitro, despite the known ATPase activity of YdiB. The estimated rate of conversion of TC-AMP by YdiBCE to t(6)A is somewhat lower than the initial rate from L-threonine, bicarbonate and ATP, which together with the stability data, is consistent with previous studies that suggest channeling of this intermediate. << Less

  Biochemistry 51:8950-8963(2012) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.