Abstract
The presence of the 7-deazaguanosine derivative archaeosine (G(+)) at position 15 in tRNA is one of the diagnostic molecular characteristics of the Archaea. The biosynthesis of this modified nucleoside is especially complex, involving the initial production of 7-cyano-7-deazaguanine (preQ(0)), an advanced precursor that is produced in a tRNA-independent portion of the biosynthesis, followed by its insertion into the tRNA by the enzyme tRNA-guanine transglycosylase (arcTGT), which replaces the target guanine base yielding preQ(0)-tRNA. The enzymes responsible for the biosynthesis of preQ(0) were recently identified, but the enzyme(s) catalyzing the conversion of preQ(0)-tRNA to G(+)-tRNA have remained elusive. Using a comparative genomics approach, we identified a protein family implicated in the late stages of archaeosine biosynthesis. Notably, this family is a paralog of arcTGT and is generally annotated as TgtA2. Structure-based alignments comparing arcTGT and TgtA2 reveal that TgtA2 lacks key arcTGT catalytic residues and contains an additional module. We constructed a Haloferax volcanii DeltatgtA2 derivative and demonstrated that tRNA from this strain lacks G(+) and instead accumulates preQ(0). We also cloned the corresponding gene from Methanocaldococcus jannaschii (mj1022) and characterized the purified recombinant enzyme. Recombinant MjTgtA2 was shown to convert preQ(0)-tRNA to G(+)-tRNA using several nitrogen sources and to do so in an ATP-independent process. This is the only example of the conversion of a nitrile to a formamidine known in biology and represents a new class of amidinotransferase chemistry.
Highlights
Duction of radical structural changes to the constituent nucleosides
It had been proposed that like other 7-deazapurines, Gϩ was derived from GTP [5,6,7], and this hypothesis was recently confirmed when GTP cyclohydrolase I was shown to be the first enzyme of the Gϩ pathway [8]. It has been known for almost a decade that 7-cyano-7-deazaguanine is a key intermediate in the pathway and is inserted into tRNA by a tRNA-guanine transglycosylase (TGT,4 EC 2.4.2.29) [9, 10], encoded by the tgtA gene and referred to as arcTGT. arcTGT is a modular enzyme comprised of an N-terminal catalytic domain that folds into an (␣/)8 barrel with a characteristic zinc-binding site and a C-terminal extension of three domains C1, C2, and C3, the last an oligonucleotide/oligosaccharide binding fold-like “PUA domain” [11] that is widely conserved in eukaryotic and archaeal RNA modification enzymes
The in vivo and in vitro data are unambiguous in establishing a role for TgtA2 in the conversion of preQ0modified tRNA to archaeosine-modified tRNA
Summary
Duction of radical structural changes to the constituent nucleosides. In archaeal tRNA, this is well illustrated by archaeosine (Gϩ, see Fig. 1A), a 7-deazaguanosinemodified nucleoside unique to Archaea [1]. It had been proposed that like other 7-deazapurines, Gϩ was derived from GTP [5,6,7], and this hypothesis was recently confirmed when GTP cyclohydrolase I was shown to be the first enzyme of the Gϩ pathway [8] It has been known for almost a decade that 7-cyano-7-deazaguanine (preQ0) is a key intermediate in the pathway and is inserted into tRNA by a tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) [9, 10], encoded by the tgtA gene and referred to as arcTGT (see Fig. 1). Using in vivo and in vitro experimental methods, we demonstrate that it encodes the final enzyme in the archaeosine biosynthetic pathway, a novel ATP-independent amidinotransferase catalyzing the direct conversion of preQ0 to archaeosine
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