Identification of Four Genes Necessary for Biosynthesis of the Modified Nucleoside Queuosine

Abstract

Queuosine (Q) is a hypermodified 7-deazaguanosine nucleoside located in the anticodon wobble position of four amino acid-specific tRNAs. In bacteria, Q is produced de novo from GTP via the 7-deazaguanosine precursor preQ 1 (7-aminoethyl 7-deazaguanine) by an uncharacterized pathway. PreQ 1 is subsequently transferred to its specific tRNA by a tRNA-guanine transglycosylase (TGT) and then further modified in situ to produce Q. Here we use comparative genomics to implicate four gene families (best exemplified by the B. subtilis operon ykvJKLM) as candidates in the preQ 1 biosynthetic pathway. Deletions were constructed in genes for each of the four orthologs in Acinetobacter. High pressure liquid chromatography analysis showed the Q nucleoside was absent from the tRNAs of each of four deletion strains. Electrospray ionization mass spectrometry confirmed the absence of Q in each mutant strain. Finally, introduction of the Bacillus subtilis ykvJKLM operon in trans complemented the Q deficiency of the two deletion mutants that were tested. Thus, the products of these four genes (named queC, -D, -E, and -F) are essential for the Q biosynthetic pathway.Nucleoside modification typically occurs in ϳ10% of the nucleosides of a particular tRNA but can involve as many as 25% of the nucleosides for a specific tRNA (1). Over 80 modified nucleosides have been characterized (1), many of which are phylogenetically conserved. The nature of nucleoside modification varies from simple methylation of the base or ribose ring to extensive "hypermodification" of the canonical bases. The latter involves multiple enzymatic steps and can result in substantive structural changes (2). Queuosine (Q) 1 is an example of a highly modified nucleoside located in the anticodon wobble position 34 of tRNAs specific for Tyr, His, Asp, and Asn (3). With few exceptions (such as yeast and mycoplasma), it is widely distributed in most prokaryotic and eukaryotic phyla (4). Q is based on a very unusual 7-deazaguanosine core, which is further modified by addition of a cyclopentendiol ring (5, 6). The Q modification has long been known and has been implicated in a number of disparate physiological phenomena, such as eukaryotic cell proliferation and differentiation (7-10), tyrosine biosynthesis (11), and bacterial virulence (12). However, despite the presence of Q in the tRNA anticodon loop, no definitive role for Q in translation has been established (13).Unlike eukaryotes that import the queuine base, bacteria synthesize Q modification de novo from a GTP starting block (14). The first stage of synthesis gives 7-cyano-7-deazaguanosine (preQ 0 ) that is then further modified into 7-aminoethyl 7-deazaguanine (preQ 1 ) (Fig. 1). The preQ 1 base is subsequently transferred to the appropriate tRNA in a guanine-exchange reaction catalyzed by a tRNA-guanine transglycosylase (TGT) (15). It is then further modified in situ to give Q (16). A second 7-deazaguanosine tRNA modification, archaeosine, is found in all archaeal tRNAs at position 15 of the D-loop (17). Archa...

Confirmation of a second natural preQ1 aptamer class in Streptococcaceae bacteria

“…Representatives of the known preQ 1 riboswitch class ) are often located upstream of a Q biosynthetic operon (Reader et al 2004), but some also have been identified upstream of other genes encoding proteins otherwise not associated with Q, and one of these gene types is classified as COG4708. Thus, we speculated that COG4708 proteins might be involved in transporting Q biosynthetic precursors.…”

Crystallization and preliminary X-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme

“…1). Using comparative genomics, QueF was identified as an enzyme family involved in Q biosynthesis (Reader et al, 2004). QueF is an NADPHdependent oxidoreductase that carries out the unprecedented reduction of a nitrile group (preQ 0 ) to a primary amine (preQ 1 ; Van Lanen et al, 2005).…”

Novel genomic island modifies DNA with 7-deazaguanine derivatives

“…1A). The first step catalyzed by GTP cyclohydrolase I (GCHI; FolE) is shared with the tetrahydrofolate synthesis pathway (10), and then three enzymes-6-carboxy-5,6,7,8-tetrahydropterin synthase (QueD), 7-carboxy-7-deazaguanine synthase (QueE), and 7-cyano-7-deazaguanine synthase (QueC)-lead to the formation of the preQ 0 moiety (11,12) (Fig. 1A).…”