Role of rare-earth elements in enhancing bioelectrocatalysis for biosensing with NAD+-dependent glutamate dehydrogenase

Abstract

Dehydrogenases (DHs) are widely explored bioelectrocatalysts in the development of enzymatic bioelectronics like biosensors and biofuel cells. However, the relatively low intrinsic reaction rates of DHs which mostly depend on diffusional coenzymes (e.g., NAD⁺) have limited their bioelectrocatalytic performance in applications such as biosensors with a high sensitivity. In this study, we find that rare-earth elements (REEs) can enhance the activity of NAD⁺-dependent glutamate dehydrogenase (GDH) toward highly sensitive electrochemical biosensing of glutamate in vivo. Electrochemical studies show that the sensitivity of the GDH-based glutamate biosensor is remarkably enhanced in the presence of REE cations (i.e., Yb³⁺, La³⁺ or Eu³⁺) in solution, of which Yb³⁺ yields the highest sensitivity increase (ca. 95%). With the potential effect of REE cations on NAD⁺ electrochemistry being ruled out, homogeneous kinetic assays by steady-state and stopped-flow spectroscopy reveal a two-fold enhancement in the intrinsic reaction rate of GDH by introducing Yb³⁺, mainly through accelerating the rate-determining NADH releasing step during the catalytic cycle. In-depth structural investigations using small angle X-ray scattering and infrared spectroscopy indicate that Yb³⁺ induces the backbone compaction of GDH and subtle β-sheet transitions in the active site, which may reduce the energetic barrier to NADH dissociation from the binding pocket as further suggested by molecular dynamics simulation. This study not only unmasks the mechanism of REE-promoted GDH kinetics but also paves a new way to highly sensitive biosensing of glutamate in vivo.

Fig. 1. Crystal structure of GDH from bovine liver (PDB: 1HWY). (a) The entire hexamer of GDH with bound NAD⁺ (represented by purple spheres) and glutamate (represented by red spheres). (b) GDH subunit consisting of the NAD⁺-binding domain, glutamate-binding domain, and connecting antenna.

Fig. 2. Effect of REEs on bioelectrocatalytic performance of NAD⁺-dependent GDH. (a) Amperometric current responses of the GDH-based biosensor toward titrations of 10 and 20 μM glutamate at 0.0 V vs. Ag/AgCl in aCSF containing 2 mM NAD⁺ in the absence or presence of 16 μM Yb³⁺, La³⁺ or Eu³⁺. (b) Relative sensitivity of the GDH-based glutamate biosensor.

Fig. 3. Steady-state GDH activity assay. (a) The absorbance of NADH at 340 nm generated in aCSF containing 0.1 mg mL⁻¹ (ca. 300 nM) GDH, 250 μM NAD⁺ and 5 mM glutamate in the absence and presence of 16 μM Yb³⁺. Linear fitting of the steady-state profile during 10–30 s yields the corresponding slope for reaction rate calculation. (b) The calculated overall reaction rate of NADH production in the absence and presence of 16 μM Yb³⁺. Error bars represent standard error of the mean (n = 3, t-test, ***P < 0.001).

Fig. 4. Transient-state GDH kinetics. (a) Typical absorbance profiles at 340 nm over 100 s showing four distinguishable phases of GDH-catalyzed glutamate oxidation in aCSF containing 0.1 mg mL⁻¹ GDH, 250 μM NAD⁺ and 5 mM glutamate in the absence (black) and presence (red) of 16 μM Yb³⁺ by stopped-flow spectroscopy. (b) Rate constants of the rate-determining NADH release in phase IV obtained by fitting the absorbance profile during 10–1500 s using two consecutive one-order kinetic modeling. Error bars represent standard error of the mean (n = 3, t-test, ***P < 0.001).

Fig. 5. Protein conformation of GDH. (a) Scattering intensities of GDH in aCSF containing NAD⁺ in the absence (black) and presence (red) of Yb³⁺ by SAXS. (b) Corresponding pair distances distribution function obtained from (a). (c) R_g encoding protein size information calculated from (b). Error bars represent standard error of the mean (n = 8, t-test, ***P < 0.001). (d) Schematic illustration of Yb³⁺-induced GDH conformation change into a more compact state.

Fig. 6. Regional changes of GDH structure. (a) ATR-FTIR spectra of GDH pre-incubated with or without Yb³⁺. Dashed frame denotes the amide I band region. (b) Fourth derivation of amide I band. (c) Compositional percentages of different GDH secondary structures by integrating peak areas of deconvoluted bands. (d) Zoomed image of GDH active site with bound NADH (purple), glutamate (cyan) and residues binding NADH (red). Lys. 126 attacking glutamate intermediate and β-sheets guarding the active site are highlighted in dark blue and light blue, respectively. (e) The most plausible anchoring site for Yb³⁺ facing three potential ligand residues, Glu. 275, Ser. 276 and Asp. 277, which constitute a β-turn near bound NADH.