High-Resolution Structure of the Nitrile Reductase QueF Combined with Molecular Simulations Provide Insight into Enzyme Mechanism

Abstract

Here, we report the 1.53-Å crystal structure of the enzyme 7-cyano-7-deazaguanine reductase (QueF) from Vibrio cholerae, which is responsible for the complete reduction of a nitrile (C≡N) bond to a primary amine (H2C–NH2). At present, this is the only example of a biological pathway that includes reduction of a nitrile bond, establishing QueF as particularly noteworthy. The structure of the QueF monomer resembles two connected ferrodoxin-like domains that assemble into dimers. Ligands identified in the crystal structure suggest the likely binding conformation of the native substrates NADPH and 7-cyano-7-deazaguanine. We also report on a series of numerical simulations that have shed light on the mechanism by which this enzyme affects the transfer of four protons (and electrons) to the 7-cyano-7-deazaguanine substrate. In particular, the simulations suggest that the initial step of the catalytic process is the formation of a covalent adduct with the residue Cys194, in agreement with previous studies. The crystal structure also suggests that two conserved residues (His233 and Asp102) play an important role in the delivery of a fourth proton to the substrate.

Structural Basis of Biological Nitrile Reduction

“…Based on molecular dynamics simulations of the bimodular QueF from V. cholerae (11), an additional conserved residue, His-233 (V. cholerae numbering), was proposed to be responsible for delivering the final proton. In the B. subtilis QueF, this residue (His-96) is not in an appropriate geometry or in close enough proximity to the thioimide to function effectively as a …”

“…3, A and B). This covalent adduct was first proposed based on spectroscopic measurements in solution (14) and later predicted from numerical simulations on the V. cholerae enzyme (11). Asp-62 from strand ␤3 forms a hydrogen bond (3.1 Å) via its O␦1 atom with the nitrogen of the thioimide (Fig.…”

“…unimodular QueF was determined at 2.5 Å using molecular replacement methods and a search model generated from the crystal structure of V. cholerae QueF (PDB ID 3BP1) (Fig. 2, A and B, and Table 1) (11). The structure of the C55A enzyme mutant was then determined by the difference Fourier method using calculated model phases from the wild-type structure ( Table 1).…”

“…Gel filtration studies of an enzyme from each subfamily, the B. subtilis and E. coli QueF, were consistent with homododecameric and dimer structures for the unimodular and bimodular enzymes, respectively (5). The latter result was surprising as it implies that the enzyme lacks the canonical T-fold quaternary architecture of all other bimodular T-fold enzymes (9,10), but recent crystallographic analysis of the bimodular enzyme from Vibrio cholerae also revealed a homodimer structure for this subfamily (11), confirming that this enzyme does lack the canonical quaternary structure of the superfamily.…”

Evidence of a sequestered imine intermediate during reduction of nitrile to amine by the nitrile reductase QueF from Escherichia coli

“…Mobile elements of the protein structure are often used to close down over the substrate while it undergoes the catalytic transformation into product. The nitrile reductase QueF deals with a notably difficult problem of intermediate sequestration (3)(4)(5)(6)(7). QueF catalyzes a four-electron reduction of a nitrile to an amine in two NADPHdependent steps, presumably via an imine intermediate.…”