Changing the Electron Acceptor Specificity of Rhodobacter capsulatus Formate Dehydrogenase from NAD+ to NADP

Abstract

Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO₂ reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO₂-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO₂ capture. Here, we investigate the nicotinamide binding site of formate dehydrogenase from Rhodobacter capsulatus for its specificity toward NAD⁺ vs. NADP⁺ reduction. Starting from the NAD⁺-specific wild-type RcFDH, key residues were exchanged to enable NADP⁺ binding on the basis of the NAD⁺-bound cryo-EM structure (PDB-ID: 6TG9). It has been observed that the lysine at position 157 (Lys¹⁵⁷) in the β-subunit of the enzyme is essential for the binding of NAD⁺. RcFDH variants that had Glu²⁵⁹ exchanged for either a positively charged or uncharged amino acid had additional activity with NADP⁺. The FdsB^L279R and FdsB^K276A variants also showed activity with NADP⁺. Kinetic parameters for all the variants were determined and tested for activity in CO₂ reduction. The variants were able to reduce CO₂ using NADPH as an electron donor in a coupled assay with phosphite dehydrogenase (PTDH), which regenerates NADPH. This makes the enzyme suitable for applications where it can be coupled with other enzymes that use NADPH.

Keywords: changing cofactor specificity; enzyme engineering; formate dehyrogenases; molybdoenzymes.

Figure 1

Schematic representation of RcFDH subunits. Subunits are indicated as follows: FdsA, red; FdsB, blue; FdsG, green; FdsD, yellow (a). Ribbon representation of the atomic model derived from the cryo-EM structure (PDB-ID: 6TG9) with subunits differentiated by the similar colors as depicted in (a). Cofactors are shown in stick presentation (b).

Figure 2

Multiple sequence alignment of FdsB subunit of RcFdh with diaphorase subunits of other organisms using COBALT from NCBI [30]. Color code for amino acids are as follows: Y,F,W = blue; R,K,H = red; G,S,T,R = yellow; V,L,M,I = green; and D,E,N,Q,A,C = no color. NAD⁺ binding site residues are marked by black arrows (a). Cartoon representation of NAD⁺ binding site of RcFdsB (PDB-ID: 6TG9) (b).

Figure 3

A total of 15% SDS-polyacrylamide gel of purified RcFDH^WT and variants after size exclusion chromatography. Samples contained 20 µg of protein (a). Molybdenum, iron, and FMN saturations of the RcFDH variants relative to RcFDH^WT as quantified by inductively coupled plasma optical emission spectroscopy (ICP-OES) and FMN quantitation, respectively. A total of 1 mol of fully saturated monomeric enzyme contains 1 mol Mo, 24 mol Fe, and 1 mol FMN that was set to a cofactor saturation of 100%, and the measured values of the purified proteins were related to that optimal saturation (b).

Figure 4

Steady-state kinetics of CO₂ reduction catalyzed by RcFDH^WT and FdsB^E259G variants using NADH (a) or NADPH (b) as the electron donor. CO₂ reduction is catalyzed by RcFDH variants using NADPH-recycling system with phosphite dehydrogenase. (c) Time-dependent formate production by RcFDH^WT and variants using NADPH as the electron donor (d). A total of 500 µL of reaction volume contained 100 mM of sodium bicarbonate, 100 µM of NADH or NADPH, 10 mM of sodium phosphite, 4 µM of phosphite dehydrogenase (PTDH), 1 µM of FMN, and 50 mM of KP_i buffer, pH 6.8. The reaction was started by the addition of 2–10 µM of RcFDH^WT or variants, and the vials were incubated for up to 18 h at room temperature. Reactions were stopped by adding 30 µL of acetone to a 100 µL sample, followed by centrifugation at 14,000× g for 10 min. The supernatant was collected and derivatized for formate quantification by GC-MS.