Mechanism of Catalysis and Substrate Binding of Epoxyqueuosine Reductase in the Biosynthetic Pathway to Queuosine-Modified tRNA

Post-transcriptional modifications at the anticodon stem-loop
of
tRNAs are key to the translation function. Metabolic pathways to these
modifications often incorporate complex enzymology. A notable example
is the hypermodified nucleoside, queuosine, found at the wobble position
of Asn, Asp, His, and Tyr encoding tRNAs. The epoxyqueuosine reductase,
QueH, catalyzes the final step in the biosynthetic pathway to queuosine.
The metalloenzyme catalyzes a two-electron reduction of epoxyqueuosine
to provide the modified tRNA. The structure of QueH from T. maritima has previously been determined and unexpectedly
contains two metal binding motifs in the active site. This includes
a predicted 4Fe-4S cluster, along with a single-metal binding site
coordinated by two cysteines along an aspartate carboxylate. In this
report, we describe the structural and biochemical analysis of the
QueH metal binding sites along with the chemistry of epoxide deoxygenation.
To probe the active-site architecture, enzyme mutants of metal binding
residues were structurally and biochemically characterized. In addition,
structural and binding experiments were used to probe interactions
of QueH with tRNA and the in vivo role of QueH and variants in Q-tRNA
synthesis was evaluated. Overall, this work provides insight into
the chemical mechanism of the final step of the queuosine biosynthetic
pathway.