The structure of glycerol trinitrate reductase NerA from Agrobacterium radiobacter reveals the molecular reason for nitro- and ene-reductase activity in OYE homologues

Abstract

In recent years, Old Yellow Enzymes (OYEs) and their homologues have found broad application in the efficient asymmetric hydrogenation of activated C=C bonds with high selectivities and yields. Members of this class of enzymes have been found in many different organisms and are rather diverse on the sequence level, with pairwise identities as low as 20 %, but they exhibit significant structural similarities with the adoption of a conserved (αβ)(8)-barrel fold. Some OYEs have been shown not only to reduce C=C double bonds, but also to be capable of reducing nitro groups in both saturated and unsaturated substrates. In order to understand this dual activity we determined and analyzed X-ray crystal structures of NerA from Agrobacterium radiobacter, both in its apo form and in complex with 4-hydroxybenzaldehyde and with 1-nitro-2-phenylpropene. These structures, together with spectroscopic studies of substrate binding to several OYEs, indicate that nitro-containing substrates can bind to OYEs in different binding modes, one of which leads to C=C double bond reduction and the other to nitro group reduction.

Scheme 1

Reactions of nitro substrates catalyzed by OYEs. Blue arrows indicate the pathway that leads to the oxazete product. Red arrows show the reaction scheme leading to the carbonyl compound.

Figure 1

Crystal structure of NerA. A) Three monomers are present in the asymmetric unit of the NerA structure. Each monomer is shown in a cartoon representation (chain A brown, chain B green, and chain C salmon) with FMN displayed as sticks and colored in yellow. B) NerA viewed from the bottom of the β-barrel, showing the β-hairpin structure typical for OYEs. C) Active site of NerA. The active site residues participating in catalysis are shown as sticks and labeled according to NerA sequence numbering. Pseudoatom centers are placed in the middles of the aromatic rings of Y65 and Y356. D) Superposition of all three NerA monomers present in the asymmetric unit in a B-factor putty representation. The loops are colored according to the three different chains (chain A brown, chain B green, and chain C salmon). The figure was prepared with PyMOL.

Figure 2

Substrate and inhibitor complexes of NerA. A) Close-up view of the structure of NerA in complexation with 4-hydroxybenzaldehyde. B) Close-up view of the NerA structure in complexation with 1-nitro-2-phenylpropene. In both cases, the 2 mF_o−DF_c electron density map before the ligands were fit into the density, countered at 1σ are shown. Important active site residues are shown as sticks and labeled by NerA sequence numbering. The figure was prepared with PyMOL.

Figure 3

Active sites of A) NerA, B) OPR-3, C) OYE-1, and D) XenA. The colored, semi-transparent surfaces each represent the shape of the associated enzyme's active site cavity. The surfaces of the cavities are colored according to the hydrophobicity/hydrophilicity of the residues lining the active site (red hydrophobic, blue hydrophilic). The small insets in the lower left corners each show a cavity representation without the surrounding enzyme. The figure was prepared with PyMOL.

Figure 4

UV/Vis spectroscopy. A) UV/Vis absorbance difference spectra at concentrations of 1-nitro-2-phenylpropene corresponding to the K_d values listed. Solid line: OYE1 (K_d=0.17 mm). Dotted line: NerA (K_d=0.73 mm). Dashed line: XenA (K_d=0.085 mm). B) UV/Vis absorbance spectrum of NerA before (dashed line) and after (solid line) titration with 1-nitro-2-phenylpropene. C) Difference spectra of NerA at increasing concentrations of 1-nitro-2-phenylpropene. D) Determination of the K_d value for the binding of 1-nitro-2-phenylpropene to NerA (0.73 mm) by a hyperbolic fit of the absorbance change at 524 nm.