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

Abstract

QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ 0 ) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.0 Å . A three-dimensional model based on homology with the tunnel-fold enzyme GTP cyclohydrolase I suggested catalysis at intersubunit interfaces and a potential role for substrate binding in quaternary structure stabilization. Guided by this insight, a second crystal form was grown that was strictly dependent on the presence of preQ 0 . This crystal form diffracted to 2.25 Å resolution.

Structural Basis of Biological Nitrile Reduction

“…Crystallization, X-ray Data Collection, and Crystal Structure Determination-Wild-type QueF and the single-site QueF mutant C55A from B. subtilis were overproduced and purified as described previously (13,14). For both the wild type and the C55A mutant, the enzyme⅐preQ 0 complex was crystallized at 293 K using the hanging drop method as described previously (13).…”

“…For both the wild type and the C55A mutant, the enzyme⅐preQ 0 complex was crystallized at 293 K using the hanging drop method as described previously (13). Briefly, the enzyme (4 mg/ml in 100 mM Tris pH 7.5, 100 mM KCl) was mixed with preQ 0 at a 1:5-1:10 molar ratio.…”

The Copper-Inducible cin Operon Encodes an Unusual Methionine-Rich Azurin-Like Protein and a Pre-Q 0 Reductase in Pseudomonas putida KT2440

“…Escape of the imine from the active site into bulk solvent could lead to conversion to the observed aldehyde. Recent homology modeling (35) suggested that B. subtilis QueF is a homodecameric protein with active sites located at intersubunit interfaces. It is possible that the affinity tag that was used in our studies to facilitate purification of CinQ interferes with the native quaternary structure of this protein and leads to the diffusion of intermediates from the active site.…”

Biosynthesis of pyrrolopyrimidines

“…QueF catalyzes covalent attachment of this Cys thiolate sidechain to the carbon atom of the cyano group of preQ 0 to from a thioimide, which is converted to preQ 0 in two successive NADPH-dependent reactions [66]. Homology modeling [67] and crystallographic structure determinations have confirmed the structural similarities between QueF and GCH I, showing that it adopts a tunnel fold and the position of the catalytically essential cysteine residue [68]. …”