Structural biology of Queuosine biosynthesis

Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5'-GUN-3' tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We have recently reported the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbours a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveal an unexpected diversity in the reductive chemistry catalyzed by these enzymes. We now wish to study the wider QueG/RdhA family from a structure/function perspective, and probe whether these enzymes can be used as new reductase catalysts, that will have distinct properties from the more traditionally used flavin/NAD(P)H based systems. We screen distinct homologues for expression and crystallization, as well as probe the existing QueG/RdhA systems by site directed mutagenesis aimed at unraveling the mechanism. The latter will be used to inform on biotechnological application with the goal to catalyse desirable reductions (such as alcohol reduction) in vitro and ultimately in vivo. A key part of the latter will be the study of the putative electron transfer partners.