Establishment of mesophilic-like catalytic properties in a thermophilic enzyme without affecting its thermal stability

Abstract

Thermophilic enzymes are generally more thermally stable but are less active at moderate temperatures than are their mesophilic counterparts. Thermophilic enzymes with improved low-temperature activity that retain their high stability would serve as useful tools for industrial processes especially when robust biocatalysts are required. Here we show an effective way to explore amino acid substitutions that enhance the low-temperature catalytic activity of a thermophilic enzyme, based on a pairwise sequence comparison of thermophilic/mesophilic enzymes. One or a combination of amino acid(s) in 3-isopropylmalate dehydrogenase from the extreme thermophile Thermus thermophilus was/were substituted by a residue(s) found in the Escherichia coli enzyme at the same position(s). The best mutant, which contained three amino acid substitutions, showed a 17-fold higher specific activity at 25 °C compared to the original wild-type enzyme while retaining high thermal stability. The kinetic and thermodynamic parameters of the mutant showed similar patterns along the reaction coordinate to those of the mesophilic enzyme. We also analyzed the residues at the substitution sites from a structural and phylogenetic point of view.

Figure 1

Relationship between the unfolding midpoint temperatures and the specific activities at 25 °C of the microbial IPMDHs on a logarithmic scale. The linear approximation of the plot for IPMDHs from Saccharomyces cerevisiae (Sc), Bacillus subtilis (Bs), Bacillus cereus (Bc), Methanothermobacter thermautotrophicus (Mt), T. thermophilus (Tt) and Sulfolobus tokodaii (St) is shown as the dotted line (correlation coefficient = 0.97). The plot of EcIPMDH (Ec) deviates from the approximation line.

Figure 2

Specific activity of TtIPMDH (magenta) and its mutants at 25 °C. Mutants that displayed improved catalytic activities at 25 °C by more than a factor of two are shown in cyan and the other mutants shown in purple. Numerical values for the mutants with improved activity are indicated above the bars. Values relative to that of TtIPMDH are indicated in parentheses.

Figure 3

Specific activities of TtIPMDH (magenta), its mutants and EcIPMDH (blue) at 25 °C. Mutants that displayed improved catalytic activities at 25 °C compared to mut#9 (gray) are shown in cyan and the other mutants shown in purple. Numerical values are indicated above the bars. Values relative to that of TtIPMDH are indicated in parentheses.

Figure 4

Free energy (a), enthalpy (b) and entropy (c) profiles along the IPMDH-catalyzed reaction coordinate at 25 °C. Colors: magenta, TtIPMDH; orange, mut9/17; green, mut9/21; blue, EcIPMDH. IS, initial state; MS, Michaelis complex state; TS, transition state.

Figure 5

Thermal melting profiles for TtIPMDH, its mutants, and EcIPMDH. Ellipticities were monitored at 222 nm. The scan rate was 1.0 °C/min. The solutions were 5 μM protein, 20 mM potassium phosphate (pH 7.6), 1 mM EDTA. Colors: magenta, TtIPMDH; orange, mut9/17; green, mut9/21; cyan, EcIPMDH.

Figure 6

(a) A phylogenetic tree built from IPMDH sequences. Thermophilic species are shown in red. (b) Amino acid residues found at positions 130, 220, 272 and 273 in the inferred ancestral amino acid sequences at nodes #1–#10 as well as in the sequences of TtIPMDH (Tt) and EcIPMDH (Ec).