Functional analyses of ancestral thioredoxins provide insights into their evolutionary history

Abstract

Thioredoxin (Trx) is a conserved, cytosolic reductase in all known organisms. The enzyme receives two electrons from NADPH via thioredoxin reductase (TrxR) and passes them on to multiple cellular reductases via disulfide exchange. Despite the ubiquity of thioredoxins in all taxa, little is known about the functions of resurrected ancestral thioredoxins in the context of a modern mesophilic organism. Here, we report on functional in vitro and in vivo analyses of seven resurrected Precambrian thioredoxins, dating back 1-4 billion years, in the Escherichia coli cytoplasm. Using synthetic gene constructs for recombinant expression of the ancestral enzymes, along with thermodynamic and kinetic assays, we show that all ancestral thioredoxins, as today's thioredoxins, exhibit strongly reducing redox potentials, suggesting that thioredoxins served as catalysts of cellular reduction reactions from the beginning of evolution, even before the oxygen catastrophe. A detailed, quantitative characterization of their interactions with the electron donor TrxR from Escherichia coli and the electron acceptor methionine sulfoxide reductase, also from E. coli, strongly hinted that thioredoxins and thioredoxin reductases co-evolved and that the promiscuity of thioredoxins toward downstream electron acceptors was maintained during evolution. In summary, our findings suggest that thioredoxins evolved high specificity for their sole electron donor TrxR while maintaining promiscuity to their multiple electron acceptors.

Keywords: electron transfer; methionine sulfoxide reductase; molecular evolution; oxidative stress; phylogenetic reconstruction; redox biology; redox homeostasis; thioredoxin; thioredoxin reductase.

Figure 1.

NADPH-dependent reductive pathways catalyzed by thioredoxin in the E. coli cytosol. Abbreviations used are as follows: thioredoxin reductase (TrxR), PDB code 1f6m (39); thioredoxin (Trx), PDB code 2trx (57); methionine sulfoxide reductase (MsrA), PDB code 1ff3 (58); PAPS reductase, PDB code 1sur (59); thiol peroxidase, PDB code 3hvs (60); ribonucleotide reductase, PDB code 3uus (61). Thioredoxin reductase, PAPS reductase, and thiol peroxidase are homodimers. For clarity, only cartoon representations of the respective monomers are shown. Ribonucleotide reductase is an α₂β₂ heterotetramer, of which only a αβ heterodimer is shown.

Figure 2.

Redox equilibria between ecTrx and the individual ancestral thioredoxins at pH 7.0 and 25 °C. Reduced ecTrx was mixed with an equimolar amount of the oxidized form of the respective ancestral Trx. After attainment of equilibrium, the reactions were quenched with acid, and all redox forms were separated in a single run by reversed-phase HPLC. Peak areas were converted to concentrations, from which redox equilibrium constants (K_eq) and redox potential (E′₀) (Table 1) were calculated according to Equations 1 and 2 (see under “Experimental procedures”), respectively. The double peaks observed for both redox forms of the Trx variants AECA, LPBCA, LAFCA, and LECA result from incomplete intracellular cleavage of the N-terminal methionine during expression of these variants (see Fig. S3 and Table S2). To confirm that equilibrium was attained, reduced ecTrx and the oxidized ancestral variants were also mixed at different molar ratios (Fig. S2). The obtained K_eq resulted to be independent of the initial mixing ratio, proving that the equilibrium was obtained.

Figure 3.

GdmCl-dependent unfolding/refolding equilibria of oxidized (blue) and reduced (red) ancestral Trxs at pH 7.0 and 25 °C. Unfolding (open symbols) and refolding (closed symbols) reactions were incubated for ≥24 h prior to the recording of the far-UV CD signal at 220 nm. Data for each unfolding/refolding equilibrium were fitted globally according to the two-state model of folding (Equation 3) and normalized (solid lines). The obtained thermodynamic stabilities of oxidized and reduced Trxs are summarized in Table 1 and Table S3.

Figure 4.

Reactivity of ancestral thioredoxins as reductants of non-natural disulfide substrates at pH 7.0 and 25 °C. a, activity of ancestral thioredoxins as catalysts of insulin (0.13 mm) reduction by DTT (1 mm). Reactions were followed by the increase in optical density at 650 nm, caused by aggregation of the reduced insulin B chain. The inverse time of aggregation onset (time required for an increase in OD₆₅₀ of 0.05) depended linearly on catalyst concentration between 0.5 and 2.0 μm Trx. The slopes of the linear regressions were defined as the specific insulin reductase activity. The last LPBCA data point at 2.0 μm Trx (in brackets) is an outlier and was not included in the linear fit. b, reduction of DTNB by the reduced Trx variants, recorded with stopped-flow kinetics under pseudo-first–order conditions (100 μm DTNB, 2 μm Trx_red) via the increase in TNB absorbance at 412 nm. Original data were fitted according to pseudo-first–order kinetics (see Table 2) and normalized (solid lines).

Figure 5.

In vitro activity of ancestral thioredoxins as electron transfer catalysts in the reconstituted NADPH-dependent reduction of S-MetO at pH 7.0 and 25 °C, catalyzed by E. coli thioredoxin reductase (TrxR) and E. coli methionine sulfoxide reductase A (MsrA). The top panel shows the complete scheme of the electron transport chain. a, specific activity of ancestral thioredoxins in NADPH-dependent S-MetO reduction. Initial velocities (V_i), recorded via the decrease in NADPH absorbance, were measured as a function of Trx concentration. Initial concentrations (0.4 mm NADPH, 4.5 μm TrxR, 4.5 μm MsrA, 10 mm S-MetO) were chosen such that V_i depended linearly on Trx concentration between 0.1 and 1 μm Trx. The slopes of the linear fits (solid lines) were defined as specific activity of the respective Trx variant in this assay. b, specific activity in NADPH-dependent S-MetO reduction of ancestral thioredoxins (proportional to the areas of the indicated squares) in the context of a phylogenetic tree. Green square, activity of ecTrx; yellow squares, Trx variants with a specific activity above 35% relative to that of ecTrx; orange squares, Trx variants with a specific activity of 10–35% relative to that of ecTrx; red crosses, Trx variants with less than 1% activity relative to that of ecTrx or no activity.

Figure 6.

Interaction between ancestral thioredoxins and E. coli TrxR (a) or E. coli MsrA (b) at pH 7.0 and 25 °C. a, determination of the catalytic parameters of the reduction of ancestral thioredoxins by NADPH, catalyzed by E. coli TrxR. Reduced thioredoxins were converted back to their oxidized state in situ with excess DTNB, and the reactions were followed via the increase in absorbance at 412 nm due to formation of 2 eq of TNB per recycled, oxidized Trx. Initial velocities (V_i) were plotted against Trx concentration. The deduced K_m and k_cat values are summarized in Table 3. Initial concentrations were 0.8 mm NADPH, 3.3 mm DTNB, and 0.04 μm TrxR, and the initial Trx concentration was varied between 0.1 and 300 μm depending on the Trx variant. The Trx variants LECA, LAFCA, and hTrx were not recognized as substrates by E. coli TrxR. b, stopped-flow tyrosine/tryptophan fluorescence kinetics of reduction of E. coli MsrA by the ancestral thioredoxins at pH 7.0 and 25 °C. The initial concentration of oxidized E. coli MsrA (1.0 μm) was kept constant, and the initial concentrations of reduced ancestral thioredoxins were varied between 5.0 and 100 μm (pseudo-first–order conditions). As an example, the normalized fluorescence increase upon MsrA reduction at initial concentrations (7 or 7.5 μm) of reduced ancestral thioredoxins is shown. The global analysis of the kinetics revealed biphasic kinetics of MsrA reduction for all ancestral thioredoxins, which were particularly evident at low excess of reduced Trx over oxidized MsrA due to a lag phase in the fluorescence trace. All data were consistent with a consecutive mechanism, with a second-order reaction (rate constant k₁) of mixed disulfide formation followed by a first-order decay (k₂) of the mixed disulfide intermediate to reduced MsrA and oxidized Trx (solid lines). The deduced rate constants k₁ and k₂ are provided in Table 3 for all Trx variants investigated.

Figure 7.

In vivo activity of ancestral thioredoxins in an E. coli trxA/metE deletion mutant under selective pressure in minimal medium with MetO as the sole source of methionine. The E. coli trxA/metE deletion strain was transformed with expression plasmids for the respective Trx variants. All plasmids had an identical plasmid background and differed only in the genetic sequence of the respective trx gene. All thioredoxin genes were under the trc promoter/lac operator control. Cells were first grown in rich medium, harvested, washed, and then resuspended with minimal medium supplemented with MetO to OD₆₀₀ of 1.0, 0.1. 0.01, 0.001, 0.0001, and 0.00001. Three 2-μl aliquots of each suspension were pipetted on an agar plate containing selective medium, ampicillin (120 μg/ml), kanamycin (50 μg/ml), and 1 mm IPTG and grown for 2 days at 25 °C. The number below each Trx variant corresponds to the ranking of its specific activity in catalyzing NADPH-dependent reduction of S-MetO in Table 3.

Figure 8.

Analysis of the interactions between ancestral thioredoxins and ecTrxR. a, structure of the ecTrxR–ecTrx complex (PDB code 1F6M (39)). The two dimers of ecTrxR are colored in two different shades of pink and the two bound E. coli thioredoxins in blue. Green, FAD molecule bound to the FAD domain. Orange, 3-aminopyridine adenine dinucleotide phosphate, analogous of NADP⁺. b, detailed view of the interface between ecTrx and ecTrxR in the X-ray structure of the ecTrx–ecTrxR mixed disulfide complex, highlighting the specific intermolecular hydrogen bonds between ecTrx Arg-73 and ecTrxR Gly-129, Arg-130, and Ala-237, and between the main chain atoms of ecTrx Ile-75 and ecTrxR Asp-139. c, activity as a substrate of ecTrxR of ancestral Trxs (see Table 3) in the context of a phylogenetic tree. Green squares, ecTrx and Trx variants with TrxR substrate efficiency >5% relative to that of ecTrx; orange squares, Trx variants with a substrate efficiency of 1–2% relative to that of ecTrx; red squares, Trx variants with less than 0.5% substrate efficiency relative to ecTrx or no activity. The two residues corresponding to the Arg-73 and Ile-75 ecTrx in the respective ancestral Trx are shown at the left corners of each square. Below the name of each variant is reported its sequence identity relative to ecTrx.