Cyclodipeptides are secondary metabolites biosynthesized by many bacteria and exhibit a wide array of biological activities. Recently, a new class of small proteins, named cyclodipeptide synthases (CDPS), which are unrelated to the typical nonribosomal peptide synthetases, was shown to generate several cyclodipeptides, using aminoacyl-tRNAs as substrates. The Mycobacterium tuberculosis CDPS, Rv2275, was found to generate cyclodityrosine through the formation of an aminoacyl-enzyme intermediate and to have a structure and oligomeric state similar to those of the class Ic aminoacyl-tRNA synthetases (aaRSs). However, the poor sequence conservation among CDPSs has raised questions about the architecture and catalytic mechanism of the identified homologs. Here we report the crystal structures of Bacillus licheniformis CDPS YvmC-Blic, in the apo form and complexed with substrate mimics, at 1.7-2.4-Å resolutions. The YvmC-Blic structure also exhibits similarity to the class Ic aaRSs catalytic domain. Our mutational analysis confirmed the importance of a set of residues for cyclodileucine formation among the conserved residues localized in the catalytic pocket. Our biochemical data indicated that YvmC-Blic binds tRNA and generates cyclodileucine as a monomer. We were also able to detect the presence of an aminoacyl-enzyme reaction intermediate, but not a dipeptide tRNA intermediate, whose existence was postulated for Rv2275. Instead, our results support a sequential catalytic mechanism for YvmC-Blic, with the successive attachment of two leucine residues on the enzyme via a conserved serine residue. Altogether, our findings suggest that all CDPS enzymes share a common aaRS-like architecture and a catalytic mechanism involving the formation of an enzyme-bound intermediate.X-ray crystallography | pulcherrimin | diketopiperazine | divergent evolution | thioesterase domain

Ribosome biogenesis requires multiple assembly factors. In Escherichia coli, deletion of RlmE, the methyltransferase responsible for the 2′-O-methyluridine modification at position 2552 (Um2552) in helix 92 of the 23S rRNA, results in slow growth and accumulation of the 45S particle. We demonstrate that the 45S particle that accumulates in ΔrlmE is a genuine precursor that can be assembled into the 50S subunit. Indeed, 50S formation from the 45S precursor could be promoted by RlmE-mediated Um2552 formation in vitro. Ribosomal protein L36 (encoded by rpmJ) was completely absent from the 45S precursor in ΔrlmE, and we observed a strong genetic interaction between rlmE and rpmJ. Structural probing of 23S rRNA and high-salt stripping of 45S components revealed that RlmE-mediated methylation promotes interdomain interactions via the association between helices 92 and 71, stabilized by the single 2′-O-methylation of Um2552, in concert with the incorporation of L36, triggering late steps of 50S subunit assembly.R ibosomes are essential ribonucleoprotein complexes that translate the genetic information encoded by mRNA into protein. Intact bacterial ribosomes, which have a sedimentation coefficient of 70S, consist of large (50S) and small (30S) subunits. Each subunit can be reconstituted in vitro from its individual component ribosomal RNAs and proteins (1-5), indicating that these components contain all of the information necessary to automatically assemble into functional subunits. Because the intermediate particles observed in an in vitro reconstitution of the subunits are similar to those observed in vivo, assembly maps that describe the order of ribosomal protein binding in vitro are useful tools for understanding ribosome biogenesis in the cell (6). However, assembly is slower in vitro than in vivo, and nonphysiological conditions, such as high-salt concentration and high temperature, are required for in vitro assembly.In the cell, ribosome assembly is coordinated with the transcription and processing of large precursor rRNAs encoded by the rrn operon (3, 7). Once transcription starts, nascent transcripts rapidly form local secondary and tertiary structures that serve as binding sites for the primary ribosomal proteins, thereby initiating ribosome assembly. However, hierarchical assembly starting at the 5′-terminal domains of the 16S and 23S rRNAs is not essential for ribosome formation in the cell (8) because nonribosomal factors, termed "assembly factors," facilitate rapid and efficient assembly of ribosomal particles. Ribosome biogenesis is an energy-consuming process in which a number of assembly factors, including RNA helicases, GTPases, protein chaperones, and rRNA-modifying enzymes, support efficient assembly of each subunit (6, 9, 10). Individual mutation or deletion of several assembly factors causes accumulation of immature 50S subunits that sediment at 40S or 45S. For example, the RNA helicase SrmB participates in the early stage of 50S assembly (11,12); deletion of srmB results in accumulation of a 40S ...

S -adenosylmethionine (SAM) is an essential metabolite and a methyl group donor in all living organisms. The intracellular SAM concentration is tightly regulated, and depletion causes hypomethylation of substrates, growth defects and pathological consequences. In the emerging field of epitranscriptomics, SAM-dependent RNA methylations play a critical role in gene expression. Herein, we analyzed the methylation status of ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) in Escherichia coli Δ mtn strain in which cellular SAM was down-regulated, and found hypomodification of several methylation sites, including 2′- O -methylation at position 2552 (Um2552) of 23S rRNA. We observed severe growth defect of the Δ mtn strain with significant accumulation of 45S ribosomal precursor harboring 23S rRNA with hypomodified Um2552. Strikingly, the growth defect was partially restored by overexpression of rlmE encoding the SAM-dependent methyltransferase responsible for Um2552. Although SAM is involved not only in rRNA methylation but also in various cellular processes, effects on ribosome biogenesis contribute substantially to the observed defects on cell proliferation.