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 NADPH-dependent reduction of 7-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.

Fig. 1.

The biosynthetic pathways to queuosine and archaeosine.

Fig. 5.

Alignment of unimodular FolE (GTP cyclohydrolase I) and YkvM sequences. For clarity and space, only sequences from select organisms are shown from among 60 sequences in the original alignment, and the N-termini have been truncated. Sequence numbers of every 10th residue are shown for E. coli FolE and B. subtilis YkvM. Secondary structure elements and nomenclature as defined by the crystal structure of E. coli FolE and by the 3D homology model of B. subtilis YkvM are shown at Upper and Lower, respectively. The conserved Cys and Glu found in the substrate binding pocket of both protein families are indicated by asterisks. The QueF motif, specific for the QueF family, is highlighted in green. The zinc binding His and Cys residues found in FolE and not in QueF are highlighted in blue. Other catalytic residues in FolE not found in QueF are highlighted in yellow. The absence of the zinc-binding and catalytic residues of FolE is the best identifier of QueF sequences in genome databases.

Fig. 2.

Activity of E. coli QueF (assays with B. subtilis QueF gave qualitatively identical results). (A) UV continuous assays of NADPH consumption. Assays were performed in 100 mM Tris·HCl (pH 7.5)/100 μM preQ₀/100 μM NADPH and monitored at 340 nm. The concentrations of enzyme are the following: ○, no enzyme; •, 19 μg/ml; ⋄, 38 μg/ml; ▪, 95 μg/ml; □, 190 μg/ml. (B) HPLC chromatogram of synthetic preQ₁ (dashed) and preQ₀ (solid). (C) HPLC chromatograms of an E. coli QueF reaction (solid) and the reaction spiked with authentic preQ₁ (dashed).

Fig. 3.

HPLC analysis of preQ₀ nucleoside. (A) HPLC chromatogram of the constituent nucleosides from digestion of preQ₀-containing 17-mer RNA. (B) HPLC chromatograms of the nucleoside components of unfractionated tRNA from wild-type Acinetobacter ADP1 (light line) and Acinetobacter ADP1 ΔykvM::sacB-Km^R (heavy line).

Fig. 4.

Primary structure organization of the YqcD and YkvM subfamilies of QueF.