Discovery and Biochemical Studies of Enzymes Involved in the Queuosine Biosynthetic Pathway

Abstract

Queuosine (Q) is a hypermodified nucleoside present at the wobble position in the 5'-GUN-3' anticodon loop of asparagine, aspartic acid, histidine, and tyrosine encoding tRNAs. This hypermodified base contains a 7-deazapurine structure common to many antibiotics, antivirals, and antineoplastic secondary metabolites. It is synthesized de novo in prokaryotes from GTP, whereas in eukaryotes it is ingested from dietary sources as the free-base queuine and exchanged for guanine in mature tRNA. Queuosine has been associated with many physiological phenomena such as cancer pathology, neoplasia, and virulence; although a discrete physiological relevance of this modification remains to be determined due to the lack of observable phenotypes associated with its respective loss. However, conservation of this modification across almost all domains of life suggests that it confers a selective advantage to its host. CPH₄ synthase (QueD) catalyzes the second step in the queuosine biosynthetic pathway entailing conversion of 7,8-dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin. By contrast, the almost structurally identical mammalian homolog catalyzes the conversion of the same substrate to 6-pyruvoyltetrahydropterin, which is an intermediate in the tetrahydrobiopterin biosynthetic pathway. Herein, we present multiple X-ray crystal structures coupled with biochemical studies that reveal an additional active site catalytic dyad in QueD responsible for the differing activity. Prior to the studies detailed in this dissertation, the enzyme responsible for catalyzing the final step in the pathway, conversion of epoxyqueuosine to queuosine, had yet to be elucidated. To search for this enzyme, we screened a library of isogenic variants of Escherichia coli where all of the nonessential genes are sequentially inactivated. RNA was extracted from each strain and analyzed using LC-MS methods, which led to the identification of a mutant strain that accumulates epoxyqueuosine and contains no queuosine. The enzyme, epoxyqueuosine reductase (QueG), has been subjected to extensive biochemical analyses both in vitro and in vivo. From these studies, we have shown QueG to contain two [4Fe-4S] clusters and cobalamin as cofactors that are absolutely required for catalysis. In addition, we have identified conserved residues that affect activity and modulate the coordination sphere around the cobalamin cofactor.