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 (CN) bond to a primary amine (H(2)C-NH(2)). 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.

Fig. 1

Queuosine biosynthetic pathway. PreQ₀ is reduced to preQ₁ by an NADPH-dependent reaction catalyzed by the enzyme QueF. Two NADPH molecules are utilized for each molecule of preQ₁ formed.

Fig. 2

Reaction mechanism. The reduction of preQ₀ (1) to preQ₁ (2) proceeds through the creation of a covalent adduct (3). Residues identified as important in the catalytic function in the text are also displayed.

Fig. 3

Crystal structure Of QueF. The protein crystallized as a tetramer (dimer of dimers). On the left, the secondary structures are colored to emphasize the ferrodoxin-like structural modules and are labeled according to the annotation in Fig. S1. A guanine (G) molecule and phosphate (PO₄) and pyrophosphate (PP_i) molecules are shown with a space-filling representation (right): carbon, green; nitrogen, blue; oxygen, red; and phosphorus, gold. On the right, the dimer structure is illustrated.

Fig. 4

Stereo view of the QueF active site (chain A). Residues interacting with the guanine substrate are depicted as thin tubes and colored according to atom type: nitrogen, blue; oxygen, red; and carbon, tan. The guanine substrate (with carbons colored green) is oriented by a number of hydrogen bonds, indicated by the broken lines. Water molecules are rendered as red spheres. Residues are labeled with their single-letter codes: D, aspartic acid; E, glutamic acid; F, phenylalanine; I, isoleucine; K, lysine; S, serine; and W, tryptophan.

Fig. 5

Simulation model of the covalent adduct. This image represents the product state of the QM/MM simulations, where the preQ₀ substrate is covalently bound to the enzyme via a thioester bond. Atom colors are the same as in the previous figure, with the addition of atom types sulfur, yellow, and hydrogen, white.

Fig. 6

Minimum energy pathway. The minimum energies for the native protein are plotted as circles for each replica (bead) from the reactant (1) to product (10) states. Energies are normalized to the reactant state. The C194S mutant results are plotted as squares.

Fig. 7

Simulation model of NADPH binding. The pocket that binds preQ₀ adapts to bind the adenine moiety of NADPH. Note that the Lys96 residue has moved to bind the 2′-phosphate group of NADPH.

Fig. 8

Simulation model of the NADPH binding pocket. The protein is represented as a surface plot with each dimer colored separately, indicating the binding cleft. NADPH and preQ₀ are represented as tubes, with the atoms colored as follows: carbon, tan; nitrogen, blue; oxygen, red; phosphorus, gold; and sulfur, yellow. Carbon atoms in preQ₀ are colored green. The adenine moiety of NADPH is bound in a pocket below the lower phosphate group.