Moritella cold-active dihydrofolate reductase: are there natural limits to optimization of catalytic efficiency at low temperature?

Abstract

Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2 degrees C or less. The enzyme is monomeric (Mr=18,291), 55% identical to its Escherichia coli counterpart, and displays Tm and denaturation enthalpy changes much lower than E. coli and Thermotoga maritima homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0 degrees C. At mesophilic temperatures the apparent Km value for dihydrofolate is 50- to 80-fold higher than for E. coli, Lactobacillus casei, and T. maritima dihydrofolate reductases, whereas the apparent Km value for NADPH, though higher, remains in the same order of magnitude. At 5 degrees C these values are not significantly modified. The enzyme is also much less sensitive than its E. coli counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency (kcat/Km) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.

FIG. 1.

Comparison of DHFR primary structures: alignment of amino acid sequences from M. profunda (MORPR), S. enterica serovar Typhimurium (SALTY), E. coli (ECOLI), Citrobacter freundii (CITFR), Enterobacter aerogenes (ENTAE), Klebsiella aerogenes (KLEAE), and Haemophilus influenzae (HAEIN) (all above 50% identity) and from T. maritima (TEMA). DHFR_Mp K20 (A19 in E. coli) and E27 (D26 in E. coli) are in bold and underlined. A dotted line above the alignment indicates the extent of the M20 loop (residues 10 to 24) as in E. coli DHFR. Secondary structures, as defined for E. coli DHFR (3) are indicated above the alignment (α-helices and β-strands). Asterisks indicate identical amino acids and dots indicate similar amino acids conserved in all eight proteins.

FIG. 2.

Effect of temperature on DHFR_Mp activity and stability. A: residual activity after 15 min incubation at the temperature indicated; assay carried out at 30°C. B: thermodependence of the activity; assay carried out for 5 min at the temperature indicated.

FIG. 3.

Stabilization of the heat-labile DHFR_Mp: heat-induced denaturation was performed in phosphate buffer (open symbols) and in phosphate buffer containing 0.1 mM. 2-mercaptoethanol and 0.1% glycerol (closed symbols).

FIG. 4.

Urea-induced unfolding curves of DHFR. The unfolded fraction of DHFR_Mp is plotted for increasing urea concentrations (closed symbols). The sigmoidal transitions of E. coli DHFR (28) and of T. maritima DHFR (6) are included for comparison. The inset shows linear extrapolation of ΔG values in the main transition region of DHFR_Mp to zero denaturant concentration.

FIG. 5.

Stability profiles of DHFRs. The Gibbs free energy of denaturation for DHFR_Mp was calculated by equation 1 with ΔH_cal = 50 kcal mol⁻¹, T_m = 45.6°C, and ΔCp = 1.5 kcal mol⁻¹ K⁻¹(upper curve) or 2.0 kcal mol⁻¹ K⁻¹(lower curve). The stability curves for E. coli DHFR are from reference for the lower curve and from reference for the upper curve. Data for T. maritima DHFR in the presence of 2.9 M guanidinium chloride are from reference . The inset shows a differential scanning calorimetry thermogram of DHFR_Mp.