Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties

Abstract

Enzymes have high catalytic efficiency and low environmental impact, and are therefore potentially useful tools for various industrial processes. Crucially, however, natural enzymes do not always have the properties required for specific processes. It may be necessary, therefore, to design, engineer, and evolve enzymes with properties that are not found in natural enzymes. In particular, the creation of enzymes that are thermally stable and catalytically active at low temperature is desirable for processes involving both high and low temperatures. In the current study, we designed two ancestral sequences of 3-isopropylmalate dehydrogenase by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus. Moreover, they had considerably higher low-temperature catalytic activity as compared with the T. thermophilus enzyme. Detailed analyses of their temperature-dependent specific activities and kinetic properties showed that the reconstructed enzymes have catalytic properties similar to those of mesophilic homologues. Collectively, our study demonstrates that ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions.

Figure 1

Phylogenetic tree used to infer ancestral IPMDH sequences. The arrow marks the node corresponding to the position of the ancestral IPMDH proteins. Red branches indicate archaeal sequences; blue branches indicate bacterial sequences. For the complete tree, see Fig. S1. The scale bar represents 0.4 substitutions per site.

Figure 2

Multiple sequence alignment of ancIPMDH-IQ, ancIPMDH-ML, T. thermophilus IPMDH, B. subtilis IPMDH, and S. cerevisiae IPMDH. Numbers above the sequences are those of the ancestral enzymes. Residues conserved among the five sequences are highlighted in green. V277 and H278, the two residues mutated in the ancestral sequences, producing ancIPMDH-IQ-VAHG and ancIPMDH-ML-VAHG, are boxed.

Figure 3

Analytical gel filtration using Superdex200 Increase resin. Proteins were applied at 5 μM in 20 mM potassium phosphate, pH 7.6, 150 mM KCl, and 1 mM EDTA with a flow rate of 0.7 ml/min. Elution positions corresponding to the dimer and hexamer are indicated. A₂₈₀, absorbance at 280 nm.

Figure 4

Thermal melting curves of the ancestral and extant IPMDHs. The change in ellipticity at 222 nm was monitored as a function of temperature. The scan rate was 1.0 °C/min. The samples comprised 5.0 μM protein in 20 mM potassium phosphate (pH 7.6), 0.5 mM EDTA. Each experiment was conducted in duplicate with identical melting profiles within experimental error. The plots were normalized with respect to the baseline of the native and denatured states. Green, ancIPMDH-IQ; orange, ancIPMDH-ML; magenta, T. thermophilus PMDH; blue, B. subtilis IPMDH; cyan, S. cerevisiae IPMDH.

Figure 5

Specific activities of the ancestral and extant IPMDHs. (a) Plot of specific activity as a function of temperature. The assay solution was composed of 50 mM HEPES (pH 8.0), 100 mM KCl, 5 mM MgCl₂, 0.2 mM D-3-IPM, 5 mM NAD⁺, and 0.1–1.0 μM protein. Each value is the average of three measurements. The specific activities of both ancIPMDH-IQ and ancIPMDH-ML were greater than that of T. thermophilus enzymes by a factor of more than three and two, respectively, below 30 °C (inset). (b) Plot of relative activity as a function of temperature. Shown are the relative values of specific activity at various temperatures compared with the activity at the optimal temperature for each enzyme. (c) Arrhenius plot of the specific activities of the ancestral and extant IPMDHs. Activation energy (E_a) was calculated from the slope of each plot. E_a: ancIPMDH-IQ, 50 kJ/mol; ancIPMDH-ML, 59 kJ/mol; T. thermophilus PMDH, 93 kJ/mol; B. subtilis IPMDH, 66 kJ/mol; S. cerevisiae IPMDH, 64 kJ/mol. Green, ancIPMDH-IQ; orange, ancIPMDH-ML; magenta, T. thermophilus PMDH; blue, B. subtilis IPMDH; cyan, S. cerevisiae IPMDH.

Figure 6

Characterization of the mutants of the ancestral IPMDHs. (a) Thermal melting curves of ancIPMDH-IQ-VAHG (blue) and ancIPMDH-ML-VAHG (magenta). The change in ellipticity at 222 nm was monitored as a function of temperature. The scan rate was 1.0 °C/min. The samples were comprised of 5.0 μM protein in 20 mM potassium phosphate (pH 7.6), 0.5 mM EDTA. Each experiment was conducted in duplicate with identical melting profiles within experimental error. The plots were normalized with respect to the baseline of the native and denatured states. (b) Plot of relative activities of ancIPMDH-IQ (green), ancIPMDH-ML (orange), ancIPMDH-IQ-VAHG (blue) and ancIPMDH-ML-VAHG (magenta) as a function of temperature. Shown are the relative values of specific activity at various temperatures compared with the activity at the optimal temperature for each enzyme.