Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U

Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.
Nucleic Acids Research (Impact Factor: 9.11). 02/2011; 39(11):4818-26. DOI: 10.1093/nar/gkr071
Source: PubMed


Transfer RNA (tRNA) contains a number of complex ‘hypermodified’ nucleosides that are essential for a number of genetic processes.
Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete
process. We propose that the modification machinery is tuned into an efficient ‘assembly line’ that performs the modification
steps at similar, or sequentially increasing, rates to avoid build-up of possibly deleterious intermediates. To investigate
this concept, we measured steady-state kinetics for the final two steps of the biosynthesis of the mnm5s2U nucleoside in Escherichia coli tRNAGlu, which are both catalysed by the bifunctional MnmC enzyme. High-performance liquid chromatography-based assays using selectively
under-modified tRNA substrates gave a Km value of 600 nM and kcat 0.34 s−1 for the first step, and Km 70 nM and kcat 0.31 s−1 for the second step. These values show that the second reaction occurs faster than the first reaction, or at a similar rate
at very high substrate concentrations. This result indicates that the enzyme is kinetically tuned to produce fully modified
mnm5(s2)U while avoiding build-up of the nm5(s2)U intermediate. The assay method developed here represents a general approach for the comparative analysis of tRNA-modifying

    • "(D) Replacing the cmo 5 U and inosine wobble nucleotides with mnm 5 U nucleotides could liberate 13 unambiguous anticodons for reassignment (33 total amino acids; changes indicated in blue). Doing so would require the inactivation cmoB [105] and the engineering of mnmE and mnmG to recognize additional tRNAs [106] [107]. Taken a step further, the maximal genetic code would have unique amino acid assignments for all NNA and NNG codons (NNA: engineer tilS [108] to lysidinylate additional tRNAs so that they only base pair with A; NNG: change the tRNA wobble bases to cytosine so that they only base pair with G). "
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    ABSTRACT: Withstanding 3.5 billion years of genetic drift, the canonical genetic code remains such a fundamental foundation for the complexity of life that it is highly conserved across all three phylogenetic domains. Genome engineering technologies are now making it possible to rationally change the genetic code, offering resistance to viruses, genetic isolation from horizontal gene transfer, and prevention of environmental escape by genetically modified organisms. We discuss the biochemical, genetic, and technological challenges that must be overcome in order to engineer the genetic code.
    No preview · Article · Sep 2015 · Journal of Molecular Biology
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    • "Therefore, MnmC2 may be required to convert nm5U34 not originating from MnmC1. Consistent with this notion are recent kinetic studies demonstrating that the activities of the MnmC1 and MnmC2 domains are independent of one another [13]. The lack of coupling between the two active sites in bifunctional MnmC likely ensures comparable turn over of nm5U34 generated from either MnmE/MnmG or MnmC1. "
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    ABSTRACT: Background Methylaminomethyl modification of uridine or 2-thiouridine (mnm5U34 or mnm5s2U34) at the wobble position of tRNAs specific for glutamate, lysine and arginine are observed in Escherichia coli and allow for specific recognition of codons ending in A or G. In the biosynthetic pathway responsible for this post-transcriptional modification, the bifunctional enzyme MnmC catalyzes the conversion of its hypermodified substrate carboxymethylaminomethyl uridine (cmnm5U34) to mnm5U34. MnmC catalyzes the flavin adenine dinucleotide (FAD)-dependent oxidative cleavage of carboxymethyl group from cmnm5U34 via an imine intermediate to generate aminomethyl uridine (nm5U34), which is subsequently methylated by S-adenosyl-L-methionine (SAM) to yield methylaminomethyl uridine (mnm5U34). Results The X-ray crystal structures of SAM/FAD-bound bifunctional MnmC from Escherichia coli and Yersinia pestis, and FAD-bound bifunctional MnmC from Yersinia pestis were determined and the catalytic functions verified in an in vitro assay. Conclusion The crystal structures of MnmC from two Gram negative bacteria reveal the overall architecture of the enzyme and the relative disposition of the two independent catalytic domains: a Rossmann-fold domain containing the SAM binding site and an FAD containing domain structurally homologous to glycine oxidase from Bacillus subtilis. The structures of MnmC also reveal the detailed atomic interactions at the interdomain interface and provide spatial restraints relevant to the overall catalytic mechanism.
    Full-text · Article · Apr 2013 · BMC Structural Biology
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    • "However, conversion of nm 5 to mnm 5 by MnmC2 is faster than conversion of cmnm 5 to nm 5 by MnmC1 [83] [86]. This kinetic behavior of the bifunctional enzyme MnmC has been proposed to be an example of biosynthetic tuning that avoids build-up of the nm 5 intermediate [86]. The nucleoside normally present at U34 of a specific tRNA should confer optimal decoding properties to it. "
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    ABSTRACT: Among all RNAs, tRNA exhibits the largest number and the widest variety of post-transcriptional modifications. Modifications within the anticodon stem loop, mainly at the wobble position and purine-37, collectively contribute to stabilize the codon-anticodon pairing, maintain the translational reading frame, facilitate the engagement of the ribosomal decoding site and enable translocation of tRNA from the A-site to the P-site of the ribosome. Modifications at the wobble uridine (U34) of tRNAs reading two degenerate codons ending in purine are complex and result from the activity of two multi-enzyme pathways, the IscS-MnmA and MnmEG pathways, which independently work on positions 2 and 5 of the U34 pyrimidine ring, respectively, and from a third pathway, controlled by TrmL (YibK), that modifies the 2'-hydroxyl group of the ribose. MnmEG is the only common pathway to all the mentioned tRNAs, and involves the GTP- and FAD-dependent activity of the MnmEG complex and, in some cases, the activity of the bifunctional enzyme MnmC. The Escherichia coli MnmEG complex catalyzes the incorporation of an aminomethyl group into the C5 atom of U34 using methylene-tetrahydrofolate and glycine or ammonium as donors. The reaction requires GTP hydrolysis, probably to assemble the active site of the enzyme or to carry out substrate recognition. Inactivation of the evolutionarily conserved MnmEG pathway produces a pleiotropic phenotype in bacteria and mitochondrial dysfunction in human cell lines. While the IscS-MnmA pathway and the MnmA-mediated thiouridylation reaction are relatively well understood, we have limited information on the reactions mediated by the MnmEG, MnmC and TrmL enzymes and on the precise role of proteins MnmE and MnmG in the MnmEG complex activity. This review summarizes the present state of knowledge on these pathways and what we still need to know, with special emphasis on the MnmEG pathway.
    Full-text · Article · Feb 2012 · Biochimie
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