Epoxyqueuosine Reductase QueH in the Biosynthetic Pathway to tRNA Queuosine Is a Unique Metalloenzyme.

Queuosine is a structurally unique and functionally important tRNA modification, widely distributed in eukaryotes and bacteria. The final step of queuosine biosynthesis is the reduction/deoxygenation of epoxyqueuosine to form the cyclopentene motif of the nucleobase. The chemistry is performed by the structurally and functionally characterized cobalamin-dependent QueG. However, the queG gene is absent from several bacteria that otherwise retain queuosine biosynthesis machinery. Members of the IPR003828 family (previously known as DUF208) have been recently identified as nonorthologous replacements of QueG, and this family was renamed QueH. Here, we present the structural characterization of QueH from Thermotoga maritima. The structure reveals an unusual active site architecture with a [4Fe-4S] metallocluster along with an adjacent coordinated iron metal. The juxtaposition of the cofactor and coordinated metal ion predicts a unique mechanism for a two-electron reduction/deoxygenation of epoxyqueuosine. To support the structural characterization, in vitro biochemical and genomic analyses are presented. Overall, this work reveals new diversity in the chemistry of iron/sulfur-dependent enzymes and novel insight into the last step of this widely conserved tRNA modification.

Discovery of novel bacterial queuine salvage enzymes and pathways in human pathogens.

Queuosine (Q) is a complex tRNA modification widespread in eukaryotes and bacteria that contributes to the efficiency and accuracy of protein synthesis. Eukaryotes are not capable of Q synthesis and rely on salvage of the queuine base (q) as a Q precursor. While many bacteria are capable of Q de novo synthesis, salvage of the prokaryotic Q precursors preQ0 and preQ1 also occurs. With the exception of Escherichia coli YhhQ, shown to transport preQ0 and preQ1, the enzymes and transporters involved in Q salvage and recycling have not been well described. We discovered and characterized 2 Q salvage pathways present in many pathogenic and commensal bacteria. The first, found in the intracellular pathogen Chlamydia trachomatis, uses YhhQ and tRNA guanine transglycosylase (TGT) homologs that have changed substrate specificities to directly salvage q, mimicking the eukaryotic pathway. The second, found in bacteria from the gut flora such as Clostridioides difficile, salvages preQ1 from q through an unprecedented reaction catalyzed by a newly defined subgroup of the radical-SAM enzyme family. The source of q can be external through transport by members of the energy-coupling factor (ECF) family or internal through hydrolysis of Q by a dedicated nucleosidase. This work reinforces the concept that hosts and members of their associated microbiota compete for the salvage of Q precursors micronutrients.