[O-]C(C([C@@H](C(*)=O)N*)SC)=O |
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Outgoing
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3-methylthioaspartate residue
(CHEBI:73599)
is a
α-amino-acid residue anion
(CHEBI:35416)
3-methylthioaspartate residue
(CHEBI:73599)
is conjugate base of
3-methylthioaspartic acid residue
(CHEBI:73619)
3-methylthioaspartate residue
(CHEBI:73599)
is substituent group from
3-methylthioaspartate(1−)
(CHEBI:73620)
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Incoming
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3-methylthioaspartic acid residue
(CHEBI:73619)
is conjugate acid of
3-methylthioaspartate residue
(CHEBI:73599)
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3-(methylsulfanyl)-L-aspartate residue
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ChEBI
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3-(methylsulfanyl)aspartate residue
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ChEBI
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3-(methylthio)-L-aspartate residue
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ChEBI
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3-(methylthio)aspartate residue
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SUBMITTER
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3-methylsulfanyl-L-aspartate residue
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UniProt
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Arragain S, Garcia-Serres R, Blondin G, Douki T, Clemancey M, Latour JM, Forouhar F, Neely H, Montelione GT, Hunt JF, Mulliez E, Fontecave M, Atta M (2010) Post-translational modification of ribosomal proteins: structural and functional characterization of RimO from Thermotoga maritima, a radical S-adenosylmethionine methylthiotransferase. The Journal of biological chemistry 285, 5792-5801 (Source: SUBMITTER) [PubMed:20007320] [show Abstract] Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826-1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 A crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family. | Lee KH, Saleh L, Anton BP, Madinger CL, Benner JS, Iwig DF, Roberts RJ, Krebs C, Booker SJ (2009) Characterization of RimO, a new member of the methylthiotransferase subclass of the radical SAM superfamily. Biochemistry 48, 10162-10174 (Source: SUBMITTER) [PubMed:19736993] [show Abstract] RimO, encoded by the yliG gene in Escherichia coli, has been recently identified in vivo as the enzyme responsible for the attachment of a methylthio group on the beta-carbon of Asp88 of the small ribosomal protein S12 [Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 1826-1831]. To date, it is the only enzyme known to catalyze methylthiolation of a protein substrate; the four other naturally occurring methylthio modifications have been observed on tRNA. All members of the methylthiotransferase (MTTase) family, to which RimO belongs, have been shown to contain the canonical CxxxCxxC motif in their primary structures that is typical of the radical S-adenosylmethionine (SAM) family of proteins. MiaB, the only characterized MTTase, and the enzyme experimentally shown to be responsible for methylthiolation of N(6)-isopentenyladenosine of tRNA in E. coli and Thermotoga maritima, has been demonstrated to harbor two distinct [4Fe-4S] clusters. Herein, we report in vitro biochemical and spectroscopic characterization of RimO. We show by analytical and spectroscopic methods that RimO, overproduced in E. coli in the presence of iron-sulfur cluster biosynthesis proteins from Azotobacter vinelandii, contains one [4Fe-4S](2+) cluster. Reconstitution of this form of RimO (RimO(rcn)) with (57)Fe and sodium sulfide results in a protein that contains two [4Fe-4S](2+) clusters, similar to MiaB. We also show by mass spectrometry that RimO(rcn) catalyzes the attachment of a methylthio group to a peptide substrate analogue that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. Kinetic analysis of this reaction shows that the activity of RimO(rcn) in the presence of the substrate analogue does not support a complete turnover. We discuss the possible requirement for an assembled ribosome for fully active RimO in vitro. Our findings are consistent with those of other enzymes that catalyze sulfur insertion, such as biotin synthase, lipoyl synthase, and MiaB. |
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