A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency

Abstract

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.

Keywords: flavoprotein; nicotinamide cofactors; protein engineering; reactive oxygen species; soluble transhydrogenase.

Figure 1

Purification and properties of the flavoenzyme soluble transhydrogenase from Escherichia coli.A, SDS-PAGE analysis of SthA purification. Lane 1, protein markers; lane 2, extract of noninduced cells; lane 3, extract of induced cells; lane 4, affinity chromatography fraction, lane 5, size-exclusion chromatography (SEC) fraction. B, size-exclusion chromatogram of purified SthA. The protein elution on a Superdex 200 10/300 column was monitored at 280 nm (blue line) and 450 nm (red line). C, absorbance spectrum of 4.0 μM SthA. D, dynamic light scattering (DLS) of the purified enzyme. The estimated radius is 8.2 ± 0.2 nm, with a molecular weight of 467.3 ± 23.0 kDa, and polydispersity of 29.1 ± 7.6%. The DLS profile is the result of three different purification batches (n = 3), while the errors as SDs are shown only above for clarity. STH, soluble transhydrogenase.

Figure 2

SthA activity and kinetics.A, buffer and pH optimum of the thioNADPH formation mediated by SthA. The enzymatic activity of 20 nM SthA was measured in the presence of 0.5 mM NADH and 0.1 mM thioNADP+ at different pH values, using 100 mM buffers (legend in the figure). The activity in 100 mM Tris at pH 8.0 was set as 100%. The data comes from biological quadruplicates (n = 4), illustrating the s.e.m. as error bars. B, effect of phosphate on the SthA-transhydrogenation. Increasing the amount of phosphate in the form of potassium phosphate (KPi as white bars) or sodium phosphate (NaPi as gray bars), we observed a decrease in the reaction rate that leveled off around 50 mM phosphate. 100% activity was fixed as the transhydrogenation rate reached at 30 °C in the reaction mixture devoid of phosphate and composed of 50 mM Tris at pH 7.5 (Activity buffer), 20 nM SthA, 0.15 mM thioNADP+, and 1.0 mM NADH (n = 3, error bars display the s.e.m.). C, observed rate of thioNADP+ reduction by SthA as function of the concentrations of the substrates NADH and thioNADP+ at 30 °C. The assays were carried out in 50 mM Tris, pH 7.5. The kinetics data in the graphs were obtained from three independent replicates (n = 3), while the error bars represent the s.e.m. D, catalytic activation in the presence of the adenine nucleotides. The reaction conditions in the absence of any activator (yellow bar) were 50 mM Tris at pH 7.5, 1.0 mM NADH, 0.15 mM thioNADP+, 20 nM SthA. The effect of ATP (pink bar), ADP (orange bar), or AMP (brown bar) was evaluated using the same reaction mixture composition, by including 5.0 mM of each nucleotide. The data comes from three independent repetitions (n = 3, the error bars illustrate the s.e.m.). STH, soluble transhydrogenase.

Figure 3

The uncoupled oxidase activity of SthA.A, scheme reaction of the uncoupling activity oxidizing NAD(P)H in the presence of dioxygen but without NAD(P)+ as electron acceptor. The reducing equivalents are initially transferred to the flavoprotein and then to molecular oxygen, generating hydrogen peroxide and superoxide anion. B, oxygen-dependent NADH oxidation mediated by SthA in the absence of NAD(P)+ as electron acceptor. In aerobic conditions (left panel), the transient reduction of the embedded FAD (red line) allows the oxidation of NADH into NAD+ (black line), with oxygen as final electron acceptor. Carrying out the same reaction anaerobically (right panel), the flavin is reduced into FADH2 and it retains the reducing equivalents without being reoxidized at the expense of O2. Both the reactions were triggered by mixing 50 μM NADH and 7.5 μM (left) or 10.0 μM (right) SthA in buffer 50 mM Tris at pH 7.5, NaCl 0.15 M, glycerol 5%. C, superoxide detection from the SthA-mediated uncoupling and transhydrogenase activities. The uncoupling oxidase activity is followed with 1.0 mM NADH (dark blue line) or 1.0 mM NADPH (light blue line). Also, the transhydrogenation between 1.0 mM NADH and 0.2 mM thioNADP+ (ocher line) leads to superoxide generation. All the reactions were carried out in biological triplicate, each of them with a single technical replicate (n = 3, s.e.m. represented as error bars), at 30 °C in 50 mM Tris at pH 7.5, 20 μM ferricytochrome c, and started by adding 600 nM SthA. D, hydrogen peroxide detection from the SthA-mediated uncoupling and transhydrogenase activities. Using the same reaction conditions employed for the superoxide measurements in Fig.3C, we followed the uncoupling activity in the presence of NADH (dark green squares) or NADPH (light green squares) at different time points of the reaction. H2O2 was also formed as the result of the transhydrogenation (red squares). The data come from independent triplicates using single technical replicates (n = 3, the error bars show the s.e.m.), while the amount of produced H2O2 was quantified for each time point in xylenol assay solution (125 μM xylenol orange, 100 mM D-sorbitol, 250 μM (NH4)2Fe(SO4)2, 25 mM H2SO4), upon incubation for 15 min at 30 °C. E, reaction rates of the ROS formation from the uncoupling activity with NADH and NADPH. The initial velocity values refer to the first minute of the reactions reported in Figure 3, C and D. The blue bars show the superoxide production (in dark blue starting from 1.0 mM NADH, in light blue from 1.0 mM NADPH), and the green bars illustrate the formed hydrogen peroxide from equimolar NADH (dark green) and NADPH (light green). STH, soluble transhydrogenase; ROS, reactive oxygen species.

Figure 4

The role of the conserved CXXXXT motif in SthA.A, a multiple sequence alignment containing STHs was built to obtain a sequence logo with the server WebLogo3 (28). Amino acids are colored according to their chemistry (polar in green, basic in blue, and hydrophobic in black). B, Alphafold-predicted structure of SthA. The FAD and NAD+ cofactors were docked based on the superimposition of the crystallographic structure of the bacterial lipoamide dehydrogenase from Thermus thermophilus HB8 (PDB entry:2EQ7). C, comparison of the initial rates of thioNADPH, superoxide, and hydrogen peroxide formation during the transhydrogenase activity of the SthA-variants. The transhydrogenation between 1.0 mM NADH and 0.15 mM thioNADP+ leads to the generation of thioNADPH (yellow bars), hydrogen peroxide (red bars), and superoxide anion (ocher bar) at different rates. The final rates represent the average of independent triplicates (n = 3), the errors report the s.e.m.. D, pre–steady-state kinetics for the reductive half-reaction of SthA mutants. The values of the reduction rate constant (kred) of the SthA variants and apparent dissociation of NADH constant (Kd) come from biological triplicates (n = 3, error bars display the s.e.m.). E, H2O2 and superoxide production from the uncoupling activity of the SthA-variants. A similar superoxide release from the oxidase activity is observed when NADH (dark blue bar) or NADPH (light blue bar) are cosubstrates. The peroxide formation displayed more significant differences among the variants, both from NADH (dark green bar) and NADPH (light green bar). The amount of uncoupled products is calculated from biological triplicates, each of them with a single technical replicate (n = 3, the error bars represent the s.e.m.), after 10 min of reaction at 30 °C including 50 mM Tris at pH 7.5, 1.0 mM NADH or NADPH, 600 nM SthA. The incubation in xylenol assay solution was carried out in the dark at at 30 °C for 15 min. STH, soluble transhydrogenase.