From cyclohydrolase to oxidoreductase: discovery of nitrile reductase activity in a common fold.
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.
Van Lanen
Steven G
SG
Department of Chemistry, Portland State University, P.O. Box 751, Portland, OR 97207, USA.
Reader
John S
JS
Swairjo
Manal A
MA
de Crécy-Lagard
Valérie
V
Lee
Bobby
B
Iwata-Reuyl
Dirk
D
D000150:Acinetobacter / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006136:GTP Cyclohydrolase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009704:Nucleoside Q / Q000096:biosynthesis; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D009704:Nucleoside Q; D010088:Oxidoreductases; D006136:GTP Cyclohydrolase