From cyclohydrolase to oxidoreductase: Discovery of nitrile reductase activity in a common fold

Abstract

The enzyme YkvM from Bacillus subtilis was identified previously along with three other enzymes (YkvJKL) in a bioinformatics search for enzymes involved in the biosynthesis of queuosine, a 7-deazaguanine modified nucleoside found in tRNA GUN of Bacteria and Eukarya. Genetic analysis of ykvJKLM mutants in Acinetobacter confirmed that each was essential for queuosine biosynthesis, and the genes were renamed queCDEF. QueF exhibits significant homology to the type I GTP cyclohydrolases characterized by FolE. Given that GTP is the precursor to queuosine and that a cyclohydrolase-like reaction was postulated as the initial step in queuosine biosynthesis, QueF was proposed to be the putative cyclohydrolase-like enzyme responsible for this reaction. We have cloned the queF genes from B. subtilis and Escherichia coli and characterized the recombinant enzymes. Contrary to the predictions based on sequence analysis, we discovered that the enzymes, in fact, catalyze a mechanistically unrelated reaction, the NADPHdependentreductionof7-cyano-7-deazaguanineto7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of queuosine. We report here in vitro and in vivo studies that demonstrate this catalytic activity, as well as preliminary biochemical and bioinformatics analysis that provide insight into the structure of this family of enzymes.tRNA ͉ modified base T he avalanche of new protein structures that have been reported over the last decade (see summary at www.rcsb. org͞pdb͞holdings.html) has made it clear that the number of scaffolds that are used to produce all of the proteins in a cell is surprisingly limited, with Ϸ80% of the proteins using one of the 400 structural folds identified to date (1, 2). Specific functions evolve by duplication, recombination, and divergence of this core repertoire (3). Analysis of the functions of the different members of a protein structural family reveal that, in general, catalytic mechanisms and chemistries are conserved in a given family whereas substrate specificity changes (4). Much rarer are the cases in which the reactions catalyzed differ among members of the family (3); the best characterized examples being the TIM barrel superfamilies (5) and the enolase superfamily (6). Understanding the molecular paths that lead to the evolution of one function from another in a given superfamily is one of the next challenges of structural biology, impacting not only our understanding of how proteins evolve, but also the task of correctly annotating the genes identified by whole-genome sequencing (7,8).We recently used comparative genomic techniques (9) to discover four previously uncharacterized bacterial genes families (queCDEF) involved in the biosynthesis of the modified nucleoside queuosine (10). Three of these families (queCDE) have homologs in Archaea and are therefore implicated in the biosynthesis of the related modified nucleoside archaeosine (Fig. 1). Both nucleosides share an unusual 7-deazaguanosine core structure but diverge in their phylogenetic distribution, location in the ...

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

“…N-terminal His 6 -tagged B. subtilis QueF (GenBank NP_389258) was overexpressed, purified and processed for His 6 -tag removal as described in Van Lanen et al (2005). Prior to use in crystallization, the enzyme was dialyzed against 100 mM Tris pH 7.5, 100 mM KCl and 2 mM dithiothreitol.…”

“…Homology between QueF and GTP cyclohydrolase I (GTP-CH-I) has been noted (Van Lanen et al, 2005) and resulted in initial functional misannotation of the QueF gene family as GTP-CH-I in genomic databases. However, we used this homology to build a threedimensional working model of B. subtilis QueF (19.4 kDa, 165 amino acids) based on the crystal structure of GTP-CH-I.…”

“…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).…”

“…Based on their amino-acid sequences, QueF family members fall into two structural subfamilies (Van Lanen et al, 2005). The YkvM subfamily is comprised of $160-amino-acid unimodular proteins with a characteristic QueF motif, i.…”

Confirmation of a second natural preQ1 aptamer class in Streptococcaceae bacteria

“…The Q modification is known to be important for translational fidelity (Bienz and Kubli 1981;Meier et al 1985;Urbonavičius et al 2001). Q biosynthesis starts with GTP and proceeds through the intermediate preQ 0 (7-cyano-7-deazaguanine) and preQ 1 (7-aminomethyl-7-deazaguanine) in a series of enzymatic steps (Kuchino et al 1976;Okada et al 1978;Iwata-Reuyl 2003;Gaur and Varshney 2005;Van Lanen et al 2005). PreQ 1 is preferentially exchanged for a specific guanine in tRNA (Noguchi et al 1982), and the remaining biosynthetic steps take place in situ (Okada et al 1979;Slany et al 1993).…”