Understanding High-Salt and Cold Adaptation of a Polyextremophilic Enzyme

Abstract

The haloarchaeon Halorubrum lacusprofundi is among the few polyextremophilic organisms capable of surviving in one of the most extreme aquatic environments on Earth, the Deep Lake of Antarctica (-18 °C to +11.5 °C and 21-28%, w/v salt content). Hence, H. lacusprofundi has been proposed as a model for biotechnology and astrobiology to investigate potential life beyond Earth. To understand the mechanisms that allow proteins to adapt to both salinity and cold, we structurally (including X-ray crystallography and molecular dynamics simulations) and functionally characterized the β-galactosidase from H. lacusprofundi (hla_bga). Recombinant hla_bga (produced in Haloferax volcanii) revealed exceptional stability, tolerating up to 4 M NaCl and up to 20% (v/v) of organic solvents. Despite being cold-adapted, hla_bga was also stable up to 60 °C. Structural analysis showed that hla_bga combined increased surface acidity (associated with halophily) with increased structural flexibility, fine-tuned on a residue level, for sustaining activity at low temperatures. The resulting blend enhanced structural flexibility at low temperatures but also limited protein movements at higher temperatures relative to mesophilic homologs. Collectively, these observations help in understanding the molecular basis of a dual psychrophilic and halophilic adaptation and suggest that such enzymes may be intrinsically stable and functional over an exceptionally large temperature range.

Keywords: X-ray crystallography; extremophiles; extremozymes; halophiles; molecular dynamics simulations; polyextremophiles; psychrophiles.

Figure 1

Effect of various parameters on the β-galactosidase stability. (A) NaCl (room temperature), (B) temperature (4 M NaCl), (C) protective effect of salt against temperature (50 °C), (D) organic solvents, 20%, v/v and 2 M NaCl (room temperature). Details are given in “Materials and Methods”. As a control, β-galactosidase without additive was used. (100% = 38,209 μmol min⁻¹ mg⁻¹).

Figure 2

Structure of hla_bga. (A) Overall structure of the hla_bga monomer shown as a ribbon model (domains A, red; B, blue; C, yellow). The modelled loop regions not visible in the electron density are shown in magenta color. (B) Superimposition of hla_bga (red) and tth_bga (green). (C) Trimeric model structure of hla_bga, the domains of one monomer is shown as in Figure 2A while the other two domains are shown in light and dark grey (D) The surface area of trimer structure of hla_bga, one monomer is colored as in Figure 2A while the other two domains are shown in light and dark grey.

Figure 3

Surface charge of various β-galactosidases. (A–I) Surface charge of the β-galactosidase from H. lacusprofundi (hla_bga) model and its structural homologs. Red and blue colors represent negative and positive charges of the protein. (A) Halorubrum lacusprofundi, hla_bga (B) Bacillus circulans sp. Alkalophilus, (PDB ID: 3TTS) (C) Bifidobacterium animalis, (PDB ID: 4UNI) (D) Bifidobacterium bifidum S17, (PDB ID: 4UZS) (E) Bifidobacterium species, (PDB ID: 5XB7) (F) Marinomonas ef1, (PDB ID: 6Y2K) (G) Rahnella sp. R3, (PDB ID: 5E9A) (H) Thermus thermophilus A4, (PDB ID: 1KWG) (I) Geobacillus stearothermophilus, (PDB ID: 4OIF). Units: kcal (mol.electron)⁻¹ (vs. salt free buffer).

Figure 4

Effect of temperature on the protein backbone root mean square deviation (RMSD) for the three simulated systems. Values are averaged over the last 20 ns of the three independent simulations for each system. Corresponding trend lines are shown with relative correlation coefficients (R2) and equations.

Figure 5

Root mean square fluctuation (RMSF) values at increasing temperatures for the two catalytic residues (evidenced by a purple shadow) and the six conserved substrate-binding residues. The RMSF was obtainted from MD simulation at (A) 10 °C, (B) 27 °C, (C) 47 °C and, (D) 72 °C.