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The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNA^{Glu, Gln, Asp}contains a variety of xnm⁵s²U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negativeEscherichia coliK12 model. Despite the ubiquitous presence of mnm⁵s²U modification, genomic analysis shows the absence ofmnmCorthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm⁵s²U to mnm⁵s²U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm⁵s²U in bothBacillus subtilisandStreptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm⁵s²U into mnm⁵s²U inB. subtilis. Analysis of tRNA modifications of bothS. mutansandStreptococcus pneumoniaeshows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm⁵s²U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm⁵s²U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications. 

Abstract Over the last 25 years, biology has entered the genomic era and is becoming a science of ‘big data’. Most interpretations of genomic analyses rely on accurate functional annotations of the proteins encoded by more than 500 000 genomes sequenced to date. By different estimates, only half the predicted sequenced proteins carry an accurate functional annotation, and this percentage varies drastically between different organismal lineages. Such a large gap in knowledge hampers all aspects of biological enterprise and, thereby, is standing in the way of genomic biology reaching its full potential. A brainstorming meeting to address this issue funded by the National Science Foundation was held during 3–4 February 2022. Bringing together data scientists, biocurators, computational biologists and experimentalists within the same venue allowed for a comprehensive assessment of the current state of functional annotations of protein families. Further, major issues that were obstructing the field were identified and discussed, which ultimately allowed for the proposal of solutions on how to move forward.