Structural and Biochemical Analysis of the Furan Aldehyde Reductase YugJ from Bacillus subtilis

Abstract

NAD(H)/NADP(H)-dependent aldehyde/alcohol oxidoreductase (AAOR) participates in a wide range of physiologically important cellular processes by reducing aldehydes or oxidizing alcohols. Among AAOR substrates, furan aldehyde is highly toxic to microorganisms. To counteract the toxic effect of furan aldehyde, some bacteria have evolved AAOR that converts furan aldehyde into a less toxic alcohol. Based on biochemical and structural analyses, we identified Bacillus subtilis YugJ as an atypical AAOR that reduces furan aldehyde. YugJ displayed high substrate specificity toward 5-hydroxymethylfurfural (HMF), a furan aldehyde, in an NADPH- and Ni²⁺-dependent manner. YugJ folds into a two-domain structure consisting of a Rossmann-like domain and an α-helical domain. YugJ interacts with NADP and Ni²⁺ using the interdomain cleft of YugJ. A comparative analysis of three YugJ structures indicated that NADP(H) binding plays a key role in modulating the interdomain dynamics of YugJ. Noticeably, a nitrate ion was found in proximity to the nicotinamide ring of NADP in the YugJ structure, and the HMF-reducing activity of YugJ was inhibited by nitrate, providing insights into the substrate-binding mode of YugJ. These findings contribute to the characterization of the YugJ-mediated furan aldehyde reduction mechanism and to the rational design of improved furan aldehyde reductases for the biofuel industry.

Keywords: 5-hydroxymethylfurfural; Bacillus subtilis; NADPH cofactor; Ni2+ cofactor; YugJ; crystal structure; furan aldehyde reductase.

Figure 1

Catalytic properties of YugJ. (a) Substrate specificity of YugJ as an aldehyde reductase. The catalytic activity of recombinant YugJ protein toward diverse aldehydes was determined in the presence of NADPH and Ni²⁺ at pH 7.4 by monitoring NADPH oxidation, which was measured by a decrease in 340 nm absorbance. (b) pH dependence of YugJ-mediated catalysis. The HMF reduction activity of YugJ was determined at different pH values in the presence of NADPH and Ni²⁺. (c) Metal dependence of YugJ-mediated catalysis. The HMF reduction activity of YugJ was determined in the absence or presence of a divalent metal ion at pH 7.4 with NADPH. (d) Temperature dependence of YugJ-mediated catalysis. The HMF reduction activity was determined at different temperatures at pH 7.4 in the presence of NADPH and Ni²⁺. (e) Catalytic efficiency of the HMF reductase YugJ derived from the Michaelis–Menten curve. The enzymatic activity of YugJ was determined at different HMF concentrations in the presence of NADPH and Ni²⁺ at pH 7.4.

Figure 2

Ni²⁺ ion binding by YugJ. (a) Overall structure of YugJ^Ni and Ni²⁺ ion coordination by YugJ residues. YugJ and Ni²⁺ in the YugJ^Ni structure are depicted as ribbons (NTD, light blue; CTD, light red) and a sphere (cyan), respectively. Ni²⁺ ion coordination by the aspartate and histidine residues of YugJ is shown in a close-up view in the right panel. (b) Structure of the YugJ^Ni dimer. One protomer is depicted as blue and red ribbons, and the other is shown as gray ribbons with gray transparent surfaces. The Ni²⁺ ions are represented by cyan spheres. (c) X-ray fluorescence spectrum collected from a YugJ crystal. The peak at 7.46 keV is derived from Ni²⁺ ions in the YugJ crystal.

Figure 3

NADP recognition by YugJ. (a) NADPH-dependent HMF reduction by YugJ. The HMF reduction activity of YugJ was determined in the presence of NADPH or NADH as a cofactor. (b) NADP-binding site of YugJ in the YugJ^NADP-NO3 structure. The NADP cofactor (carbon, green sphere; nitrogen, blue sphere; oxygen, red sphere; phosphorus, orange sphere; interatomic bond, green stick) and its neighboring nitrate ion (nitrogen, blue sphere; oxygen, red sphere; interatomic bond, yellow stick) are represented by ball-and-stick models. The NADP-binding residues of YugJ are shown as gray lines on the YugJ structure (gray ribbons). In particular, the YugJ residues that form polar interactions with NADP are depicted as gray sticks. (c) NADP binding-mediated restriction of YugJ interdomain flexibility. The YugJ^Ni (chain A, green ribbons; chain B, magenta ribbons), YugJ^NADP-NO3 (cyan ribbons), and YugJ^NADP-Ni (orange ribbons) structures are superimposed based on their CTDs.

Figure 4

Nitrate binding of YugJ and inhibitory activity of nitrate on YugJ catalysis. (a) Putative substrate-binding site of YugJ at or near the nitrate ion in the YugJ^NADP-NO3 structure. YugJ is depicted as gray ribbons. The NADP cofactor and nitrate ion are shown as ball-and-stick models, as shown in Figure 3b. The electron density of the nitrate ion (3σ in the Fo−Fc omit map) is represented by cyan meshes. The nitrate-binding and metal-coordinating residues of YugJ are shown as gray sticks on the YugJ structure. The hydrogen bonds of the nitrate ion with YugJ residues and NADP are represented by black dotted lines. (b) Nitrate-specific inhibition of YugJ activity. The HMF reduction activity of YugJ was determined in the presence of lithium nitrate or lithium sulfate. (c) Closed state of the α9–α10 loop lid in the YugJ^NADP-NO3 structure (magenta ribbons) in contrast to an open state in the YugJ^NADP-Ni (cyan ribbons) and YugJ^Ni (green ribbons) structures.

Figure 5

YugJ-HMF complex model based on molecular docking. HMF was docked on the YugJ-NADP-Ni²⁺ model. HMF and NADP are represented by light blue and green ball-and-stick models, respectively. The Ni²⁺ ion is represented by a cyan sphere. The HMF-binding residues and Ni²⁺-coordinating residues of YugJ are shown as gray sticks on the YugJ structure (gray ribbons). The hydrogen bonds of HMF with YugJ residues are represented by black dotted lines. The aldehyde and hydroxyl groups of HMF are indicated by blue and red dotted boxes, respectively.

Figure 6

A model for YugJ binding to NADPH and HMF. The YugJ NTD and CTD are shown as blue and red ribbons, respectively, and the α9–α10 loop lid in the CTD is highlighted in yellow. The NADPH and Ni²⁺ cofactors are depicted as green sticks and a cyan sphere, respectively. NADPH and HMF binding-induced structural changes in YugJ are represented by thick red arrows.