Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase

Significance

Combining comparative genomics, mutagenesis, kinetic analysis, and molecular modeling provides a powerful way to explore and understand the structure and function of proteins under extreme and potentially astrobiological conditions. Alignment of closely related cold-active and mesophilic β-galactosidase enzymes from halophilic Archaea, followed by mutagenesis and kinetic analysis, demonstrates the importance of specific amino acid residues in temperature-dependent catalytic activity, while molecular modeling provides a structural framework for their mechanism of action. Such an interdisciplinary approach shows how a very small fraction of conserved residues that are divergent from mesophilic homologs are key to enhancing catalytic activity at cold temperatures and underscores the power of combining genomics and genetics with biochemistry and structural biology for understanding polyextremophilic enzyme function.

Abstract

The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic β-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or β-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-β-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (k_cat/K_m) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both K_m and k_cat (three substitutions) or either K_m or k_cat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme.

Halorubrum lacusprofundi, a halophilic Archaeon, is able to survive and grow in extremely low-temperature and high-salinity conditions (1, 2). Its natural habitat, Deep Lake in the Vestfold Hills of Antarctica, is notable for perennially cold temperatures (+11.5 to −18 °C) and high (28% wt/vol) thalassic salinity, with a dominance of NaCl. Remarkably, Deep Lake never freezes even at the lowest temperatures encountered in its environment, due to salinity-dependent freezing point depression. H. lacusprofundi growth has been recorded down to −1 °C and its survival demonstrated after trips to Earth’s cold stratosphere with freezing-thawing, making this species of considerable interest from an astrobiology standpoint (3). The molecular basis for its survival is relevant to the search for life on Mars, as well as on the many newly discovered planets in our galaxy (4–6).

The genome sequence of H. lacusprofundi includes a carbohydrate utilization gene cluster important for survival in its extreme oligotrophic environment, including a bga gene (Hlac_2868) which encodes a family 42 β-galactosidase (7). Family 42 β-galactosidases are usually found in extremophiles and catalyze hydrolysis of terminal nonreducing β-d-galactose residues in β-d-galactosides (EC 3.2.1.23). The β-galactosidase of H. lacusprofundi most likely functions to break down even trace levels of carbohydrates that may be present in the Antarctic environment (1). The enzyme has optimal activity in the 2–4 M NaCl range, retaining measurable activity at temperatures as low as 0 °C (7). The considerable volume of work on β-galactosidases historically makes them excellent models (8). As a result, H. lacusprofundi β-galactosidase serves as an ideal model enzyme for low-temperature studies (9).

To explore the H. lacusprofundi β-galactosidase structure and function, we constructed a homology model of the enzyme using the closely related thermophilic homolog found in Thermus thermophilus (10, 11) (Fig. 1). The T. thermophilus β-galactosidase contains a TIM barrel with eight repeats of βα units, and E141 (β-strand 5) and E312 (β-strand 8) serving as the catalytic residues (10). Based on the homology between the halophilic and thermophilic protein sequences, the TIM barrel active residues in the H. lacusprofundi protein have been identified as E142 and E312 (12). The haloarchaeal enzyme is distinguished by highly acidic character typical of halophilic proteins, with significantly greater predicted surface charge (net −65) compared with the nearly neutral T. thermophilus enzyme (net −4) (13, 14).

Fig. 1.

Ribbon diagram of H. lacusprofundi β-galactosidase family 42 monomer homology model. Domains A (light blue, with β sheets of the TIM barrel highlighted in dark blue), B (yellow), and C (red) are differentiated by color. The two active site residues (E142 and E312) within the TIM barrel loops are shown in stick form and labeled in black. Residues targeted for mutation (N251, A263, I299, F387, I476, and V482) are highlighted in green, labeled in red, and indicated by an arrowhead.

The H. lacusprofundi β-galactosidase sequence was included in a genome-wide analysis of >600 haloarchaeal proteins from closely related mesophilic Haloarchaea (12). Similar amino acid differences were observed for the β-galactosidases as for other conserved haloarchaeal orthologous proteins, with substitutions in <8% of positions invariant in homologous proteins from the mesophilic halophiles. These variations found in H. lacusprofundi β-galactosidase and other proteins from the cold-adapted species result in reduced surface negativity, increased hydrophobicity, and relatively subtle changes to sizes of buried polar, nonpolar, and aromatic residues conserved in the mesophiles, making it an ideal model for deeper analysis (12).

In the present study, we addressed the question of cold adaptation experimentally, selecting six amino acid residues in the H. lacusprofundi β-galactosidase enzyme for mutagenesis that comparative genomics predicted as being important for cold activity. Through expression, purification, kinetic analysis, and modeling, this study of a cold-adapted polyextremophilic enzyme confirmed the importance of the substituted residues in enzyme function at cold temperatures, and provided kinetic and structural insights into the basis for its psychrophilic character.

Results

Identification and Mutagenesis of Key β-Galactosidase Amino Acid Residues.

To identify amino acid residues in H. lacusprofundi β-galactosidase that play important roles in enzyme activity in cold temperatures, we aligned the eight closely related haloarchaeal family 42 glycoside hydrolases available in the CAZy database website (www.CAZy.org) (15) using ClustalX version 2.1 (16). The cold-adapted H. lacusprofundi enzyme sequence was compared with homologs from the following mesophilic haloarchaea: Haloferax lucentense, Haloferax volcanii, Halopiger xanaduensis, Halorhabdus tiamatea, Halostagnicola larsenii, Haloterrigena turkmenica, and Natronococcus occultus. The analysis identified six unclustered amino acids that were uniquely diverged in the H. lacusprofundi enzyme while being completely conserved in the mesophilic species (Fig. S1 and Table S1).

The six diverged amino acid residues in H. lacusprofundi β-galactosidase were targeted for substitution with conserved residues in the mesophilic enzymes by site-directed mutagenesis. For this purpose, we first placed the H. lacusprofundi bga gene under control of the cold-inducible cspD2 promoter in Halobacterium sp. NRC-1, a genetically tractable haloarchaeon lacking β-galactosidase (7). We next inserted a His-tag in the bga gene immediately downstream of its start codon to construct plasmid pRK42 and facilitate purification of the expressed proteins (Fig. 2, Left). Restriction fragments harboring N251D, A263S, I299L, F387L, I476V, and V482L substitutions were designed, synthesized, and used to replace the corresponding regions in plasmid pRK42. The wild-type and mutated bga genes on pRK42 derivatives (Table S2) were then transformed into the expression host (7).

Fig. 2.

Expression of wild-type and mutated β-galactosidases in Halobacterium sp. NRC-1. (Left) Expression vector showing the location and transcriptional orientation of bla (β-lactamase for ampicillin resistance), mev^r (HMG-CoA reductase for mevinolin resistance), bga [β-galactosidase, His-tag (red *)], and rep (the Halobacterium pGRB replicase gene). The cspD2 promoter (P_cspD2) is indicated by a thin black arrow, and restriction sites within the bga gene used for mutagenesis are labeled AgeI, RsrII, PvuII, BstXI, and BamHI. (Right) Profiles of native polyacrylamide gels showing the electrophoretic mobility of wild-type β-galactosidase (A) and six mutated enzymes: N251D (B), A263S (C), I299L (D), F387L (E), I476V (F), and V482L (G). The molecular weight markers shown are 150-kDa alcohol dehydrogenase (MW1) and 66-kDa albumin (MW2).

Halobacterium sp. NRC-1 transformants expressing the wild-type and mutated bga genes were used for enzyme production. For purification of each enzyme, cultures were grown at 42 °C, induced at 15 °C, and subjected to hypotonic cell lysis and nickel affinity chromatography. After β-galactosidase activity was determined colorimetrically using o-nitrophenyl-β-galactoside (ONPG) as substrate, active fractions were combined and aliquots analyzed by SDS/PAGE. A strong Coomassie blue-stained band was observed for each enzyme, with an apparent molecular weight of ∼100 kDa ∼30% larger than the predicted 78 kDa, as previously observed for this and other halophilic proteins due to their acidic nature and high negative charge (7). Activity of the purified enzymes was subsequently determined by native gel electrophoresis and an in situ enzymatic activity assay using the fluorescent substrate 4-methylumbelliferyl β-d-galactopyranoside (MUG). In each case, a single prominent fluorescent band was identified with identical mobility to the wild type (Fig. 2, Right), consistent with an enzymatically active monomeric protein.

Steady-State Kinetic Analysis of Wild-Type and Mutated β-Galactosidases.

We performed steady-state kinetic analysis of purified wild-type and mutated β-galactosidases at temperatures from 0 to 25 °C in 5 °C increments using ONPG as substrate. All reactions were performed at previously determined optimal ionic conditions (2 M KCl) in triplicate (7), followed by determination of the Michaelis constant (K_m), catalytic rate constant (k_cat), and catalytic efficiency (k_cat/K_m) using Shimadzu UVProbe software. We compared the kinetic constants and catalytic efficiency from the wild-type and mutated enzymes to identify any changes in activity that may have resulted from the substitutions (17) (Fig. 3).

Fig. 3.

Steady-state kinetics of wild-type and mutated β-galactosidases. Plots of the Michaelis constant (K_m, mM ONPG; Top, y-axis), catalytic rate constant (k_cat, s⁻¹; Middle, y-axis), and catalytic efficiency (k_cat/K_m, s⁻¹ × mM ONPG⁻¹; Bottom, y-axis) for the wild-type (blue) and each mutated enzyme (red) are shown over the temperature range of 0–25 °C on the x-axis, with SEs shown as error bars. The mutation is indicated in the header of each graph. Semilog plots for k_cat and k_cat/K_m are shown in Fig. S2, and statistical data are provided in Tables S4 and S5.

Consistent with our predictions, all six mutations resulted in changes to the K_m, k_cat, and k_cat/K_m values in a temperature-dependent manner. Four substitutions (N251D, I299L, F387L, and V482L) resulted in the expected increase in K_m values with reduced temperatures and reversal of the slope compared with the wild type (Fig. 3, Top). In contrast, A263S exhibited increased K_m levels throughout the temperature range, and I476V led to K_m values nearly identical to those of the wild type. For k_cat, four substitutions (N251D, I299L, I476V, and V482L) yielded faster rates with higher temperatures and slightly slower or similar rates at low temperatures, while two (A263S and F387L) exhibited nearly identical or slightly slower rates throughout (Fig. 3, Middle and Fig. S2). These combined effects reduced catalytic efficiency at lower temperatures and enhanced catalytic efficiency at higher temperatures for nearly all of the mutated enzymes, A263S excepted (Fig. 3 and Fig. S2).

N251D.

The K_m value of the N251D enzyme compared with the wild type was higher from 0 to 5 °C and plateaued at values slightly lower than that of the wild type at temperatures ≥10 °C (Fig. 3, Top). The k_cat values were nearly identical at 0 and 5 °C and increased progressively from 10 to 25 °C compared with the wild type (Fig. 3, Middle). As a result, the N251D enzyme exhibited slightly decreased catalytic efficiency than the wild type at temperatures of 0–5 °C (approximately twofold reduced at 0 °C) and progressively increased catalytic efficiencies at temperatures ≥10 °C (Fig. 3, Bottom). These findings are consistent with the N251D substitution resulting in reduced relative activity at the lowest temperatures primarily from K_m effects and increased activity at higher temperatures primarily from k_cat effects.

A263S.

The effects of the A263S mutation were unusual, in that the K_m values were consistently higher and the k_cat values lower than those of the wild type over the entire temperature range (Fig. 3, Top and Middle). Thus, the catalytic efficiency for A263S was reduced compared with the wild type at all temperatures, with slightly reduced effects at lower temperatures and slightly increased effects at higher temperatures (Fig. 3, Bottom). These findings indicate that the A263S mutation has deleterious effects on K_m and k_cat, which may result from the proximity of the substitution to the active site, with very slightly increased activity at higher temperatures.

I299L.

The K_m value of I299L vs. temperature was of opposite slope compared with that of the wild type, with higher values from 0 to 15 °C and lower values from 20 to 25 °C (Fig. 3, Top). The k_cat value was higher than that of the wild type at all temperatures, but the difference was enhanced at higher temperatures (Fig. 3, Middle). The catalytic efficiency for I299L was progressively higher than that of the wild type with increasing temperature except at 0 °C, where catalytic efficiency was slightly reduced compared with the wild type (Fig. 3, Bottom). These effects resulted from a combination of changes in both K_m and k_cat.

F387L.

The K_m value of F387L was higher than that of the wild type at lower temperatures (0–15 °C), and nearly identical from 20 to 25 °C (Fig. 3, Top). The k_cat of F387L was slightly higher than that of the wild type at all temperatures, with no difference observed over the temperature range (Fig. 3, Middle). The catalytic efficiency of F387L was slightly lower than that of the wild type at the lowest temperatures (0–5 °C) but slightly greater at higher temperatures (20–25 °C), indicating a less active enzyme at low temperatures and a more active enzyme at higher temperatures, due largely to the K_m effects (Fig. 3, Bottom).

I476V.

There was little difference in the K_m values between I476V and the wild type over the entire temperature range (Fig. 3, Top). The k_cat value was also unchanged at the lowest temperatures (0–5 °C), but increased progressively from 10 to 25 °C (Fig. 3, Middle). Consequently, due primarily to the increase in k_cat, I476V catalytic efficiency was progressively higher than that of the wild type above 10 °C, while being nearly identical at lower temperatures (Fig. 3, Bottom). Therefore, I476V conformed to the expected change in temperature activity, albeit only at the more moderate temperatures above 10 °C.

V482L.

The V482L enzyme had progressively greater K_m at reduced temperatures compared with the wild type over the entire 0–25 °C range (Fig. 3, Top). Compared with the wild type, the k_cat for V482L was slightly lower from 0 to 5 °C and slightly higher at higher temperatures (Fig. 3, Middle). The resulting catalytic efficiency for V482L was lower from 0 to 15 °C, approximately twofold reduced at 0 °C, and higher at 25 °C (Fig. 3, Bottom). These findings show that the V482L substitution reduces catalytic activity at the lower temperatures and increases it at the highest temperature, due to the combined effects of K_m and k_cat.

Modeling of β-Galactosidase.

We next addressed the structural effects of the six mutated residues in the H. lacusprofundi β-galactosidase using Swiss-PDBViewer (11). Based on our previously constructed homology model (12), four of the six substitutions were largely within buried regions (A263 and I299 were <5% surface accessible, V482 was ∼10% surface accessible, and N251 was ∼15% surface accessible), while two substitutions were in the more surface-accessible residues F387 (∼35%) and I476 (∼30%) (Fig. 1 and Table S1). The six amino acid residues were substituted and compared with the wild-type enzyme by neighbor, surface, and rotamer analysis to predict changes to charges, H-bonds, and packing.

Three β-galactosidase mutations in buried residues—N251D, I299L, and V482L—exhibited the expected temperature effects (Fig. 1 and Table S1). The N251 residue was located in the ninth α-helix surrounding the TIM-barrel structure, 18–20 Å away from the E142/312 active site residues (Fig. 1). Substitution of N251 with the D residue in mesophiles resulted in increased negative charge and likely electrostatic repulsion with D254, while maintaining H-bonding to S216 (Fig. 4A). I299 was located in the tenth α-helix surrounding the TIM barrel, ∼16 Å from the active site (Fig. 1). When substituted with the conserved L residue from the mesophilic enzyme, the packing around Y253 appeared to be perturbed compared with I in the wild-type H. lacusprofundi enzyme (Fig. 4C). V482 was located within a β-sheet in domain B, 33 Å from the active site E142/312 residues (Fig. 1). Substitution of V482 with the mesophilic L residue inserted a slightly larger side chain within the nearby coiled region (Fig. 4F), likely perturbing packing. The fourth buried residue, A263, located within the sixth β-sheet of the TIM barrel, was only 9–10 Å away from the active site side chains (Fig. 1). Substitution with an S residue likely resulted in an additional H-bond with T240, also part of the TIM barrel. The new H-bond may result in decreased catalytic efficiency (Fig. 4B).

Fig. 4.

Models of wild-type and mutated β-galactosidases. The most likely rotamers of six amino acids—N251D (A), A263S (B), I299L (C), F387L (D), I476V (E), and V482L (F)—are shown with wild-type residues (Left) and mutated residues (Right). Surrounding amino acid residues within 4 Å are included, with each residue labeled with its single-letter amino acid code and residue number, with substituted residues in pink. The arrow indicates surface exposure. Blue represents nitrogen; red, oxygen; yellow, sulfur; and gray, carbon. Hydrogen bonds are shown in green.

Mutations F387L and I476V, both of which were partly surface-exposed, exhibited the expected temperature effects. The F387 residue was located 19–20 Å from the active site residues within the fourteenth helix surrounding the TIM barrel (Fig. 1). Residue I476 was located in a coiled region of domain B ∼25 Å from the active site residues (Fig. 1). When F387 was substituted with L and I476 was substituted with V, both amino acid packing and the hydration shell around the protein were potentially perturbed, with a greater effect of the former due to the loss of a surface-exposed aromatic group (Fig. 4 D and E). Interestingly, the temperature effect of the F387L substitution was primarily on the Michaelis constant, whereas that of the I476V substitution was on the catalytic rate.

Discussion

We investigated the β-galactosidase enzyme of H. lacusprofundi to determine which amino acid residues are key to the function of a polyextremophilic protein at cold temperatures. Alignment of amino acid sequences from cold-adapted species with homologs from closely related mesophilic haloarchaea identified six residues potentially important for cold activity. Our modeling showed that four of the six diverged residues were dispersed around the TIM barrel of domain A containing the active site, while two were located within domain B, far from the active site. When these residues were substituted with amino acids conserved in mesophilic homologs, the Michaelis and/or the catalytic rate constant(s) were perturbed in a temperature-dependent manner, with resulting effects on catalytic efficiency. Five of the six mutations clearly exhibited the expected temperature effects of reduced catalytic activity at lower and/or increased catalytic activity at higher temperatures, with only one mutation showing reduced activity at all temperatures. Modeling suggested that the substitutions alter internal charge, packing, or H-bonding of the H. lacusprofundi enzyme, consistent with predictions from our previous comparative genomic analysis, and also affects interactions with the hydration shell (18, 19).

While representing one of the first detailed experimental studies of a polyextremophilic enzyme, our findings show better catalytic efficiency (k_cat/K_m) for H. lacusprofundi β-galactosidase at reduced temperature compared with the mutated variants, extending our conceptual understanding of psychrophilic enzymes (18, 19). In four of the mutated β-galactosidase enzymes—N251D, I299L, F387L, and V482L— primary temperature effects were observed for K_m, with increases at colder temperatures and reversal of the slope compared with the wild type (Fig. 3). The mutated enzymes displayed decreased substrate binding and reaction velocity at colder temperatures. Three of these substitutions—N251D, I299L, and V482L—as well as I476V, exhibited decreased k_cat at colder temperatures and/or increased k_cat at warmer temperatures. Only A263S showed decreases in catalytic efficiency over the entire temperature range, resulting from increased K_m and decreased k_cat. Together, mutation of five of the six diverged residues in β-galactosidase clearly showed the expected decreases in catalytic efficiency at low temperatures. Three of these resulted from changes in both K_m and k_cat, while the other two resulted from changes in either K_m or k_cat alone. Therefore, depending on the substitution, either or both substrate binding and the rate of catalysis may be improved in the cold-active enzyme.

Although few structural studies are available to date, psychrophilic enzymes are thought to optimize activity in cold temperatures by increasing their conformational flexibility (19, 20). The H. lacusprofundi β-galactosidase model provides a valuable and unique context for visualizing structural perturbations resulting from the amino acid substitutions that may account for conformational flexibility (Fig. 4). Among the four substitutions in buried amino acid residues, an additional charge (N251D) and H-bond (A263S) were observed, as were conservative changes (I299L and V482L). For N251D, a negative charge was added but did not result in formation of any new ion pairs. For A263S, the substitution resulted in an additional hydroxyl group and H-bond (21). For I299L and V482L, the substitutions likely subtly affected the packing of small hydrophobic side chains. All of these substitutions likely led to strengthening of the internal packing of amino acids and increased intramolecular forces stabilizing the enzyme (18).

Cold-adapted proteins of haloarchaea also have been found to have reduced surface charges, resulting in looser binding of water molecules in their hydration shell (13, 22). Looser binding of water would lead to greater flexibility in the protein and better function in colder temperatures. Two substitutions, F387L and I476V, were substantially more surface-accessible (∼30–35%) in the enzyme model and may have affected the hydration shell along with altering packing. Interestingly, kinetic analysis showed that while only K_m was changed for F387L, k_cat was solely affected in I476V. These observations likely reflect differences in hydration potential changes for the two substitutions, which may lead to opposite effects (23). The substantial difference in the distances of the mutated residues to the active site of β-galactosidase (>30 Å for I476V) suggest that the structural effects may be transmitted over relatively large distances in the β-galactosidase protein (24).

Genomic analysis has shown that haloarchaeal proteins are generally negatively supercharged, with a proteome-wide unimodal distribution of isoelectric points with a mode of ∼4, a hallmark of these species (13, 25). This remarkable property is responsible for mutual repulsion, increasing protein solubility and preventing aggregation in the highly saline conditions of their cytoplasm. The H. lacusprofundi β-galactosidase enzyme is typical of such proteins, possessing considerably more negative charges in contrast to the nearly neutral T. thermophilus homolog (7, 12). Interestingly, we found by native gel analysis that the haloarchaeal enzyme is monomeric as opposed to the trimeric form for the thermophile, consistent with increased solubility and mutual repulsion observed for halophilic proteins (10). The T. thermophilus protein was also initially reported to be a monomer, possibly reflecting different purification methods and protein concentrations used in the subsequent crystallographic studies (26).

We know of no other studies that have used such a combined approach (including genomics/mutagenesis/kinetics/modeling) to address the general properties of cold-active enzymes (27). One of the challenges in such genome-wide studies has been in discriminating phylogenetic effects from biochemical factors. In the case of halophilic Archaea, which form a tight phylogenetic clade, the availability of a cold-adapted species from a perennially cold environment that is closely related to a set of mesophiles from temperate habitats provided an ideal dataset for our comparative analysis (12). We tested the amino acid changes in H. lacusprofundi β-galactosidase compared with the mesophilic haloarchaea, including A to S, I to L, I to V, and V to L, which were among the most common substitutions in our genome-wide analysis (12). These changes represented a considerably higher fraction (1.3–2.6%) of cold substitutions compared with the average (∼0.3%). These observations highlight the importance of our finding of a very small number of amino acid residues substituted (<1%) in shifting the enzyme activity toward cold temperatures.

The remarkable polyextremophilic properties of H. lacusprofundi makes it of special interest for astrobiology (3). An understanding of the cold activity of β-galactosidase is particularly important in the search for life on other planets by extending the range of potential habitability to colder conditions, even below 0 °C, due to the reduced freezing point at high NaCl concentrations. Such cold-adapted enzymes are also likely to be less susceptible to cold denaturation, and are apt to maintain activity at high concentrations of salts and even in organic-aqueous solvent mixtures (7). These unusual properties together also make the H. lacusprofundi β-galactosidase enzyme of special interest for biotechnology (13, 22). The results presented here confirm that divergence of a small number of evolutionarily conserved mesophilic haloarchaeal amino acid residues in the H. lacusprofundi β-galactosidase enzyme is key to its cold adaptation in high-NaCl conditions and provides a framework for a deeper understanding of the function of polyextremophilic enzymes generally.

Materials and Methods

Mutagenesis and Expression of H. lacusprofundi β-Galactosidase.

For mutagenesis and expression of H. lacusprofundi β-galactosidase, we used the expression plasmid pRK42, a derivative of the previously described Halobacterium expression plasmid pMC2 (7). In pRK42, a His-tag was cloned into the NdeI restriction site upstream of the coding region of the bga gene of pMC2. Four restriction sites—AgeI, PvuII, BstXI, and RsrII—were identified in the bga gene for partial gene synthesis and replacement of the wild-type gene in pRK42 (Fig. 2, Left). The H. lacusprofundi bga gene restriction fragments containing mutations were synthesized by GeneArt (www.lifetechnologies.com) and cloned into pMA vectors, followed by digestion and cloning in pRK42. AgeI-PvuII restriction fragments of 355 bp were synthesized for bga mutant derivatives N251D (AAC → GAC) and A263S (GCC → TCG), and PvuII-BstXI restriction fragments of 333 bp were synthesized for bga mutant derivatives I299L (ATC → CTC) and F387L (TTC → CTG), and cloned to replace the corresponding fragments in pRK42 (Table S2). For I476V (ATC → GTC) and V482L (GTT → CTC), due to the presence of an additional RsrII site in the bga gene, a three-way ligation was used for construction, by digestion of pRK42 with AgeI (which overlaps one RsrII site) and RsrII purification of two backbone fragments, AgeI-RsrII (6.9 kb) and AgeI-BstXI (688 bp), and ligation with the synthetic BstXI-RsrII restriction fragment of 288 bp.

The different pRK42 constructs were transformed into Escherichia coli DH5α and verified by restriction digestion analysis, PCR amplification, and sequencing using the primers listed in Table S3. The pRK42 constructs were transformed into Halobacterium sp. NRC-1 using a standard PEG/EDTA method (28). Transformants were selected on CM⁺ agar plates supplemented with 20 µg/mL mevinolin and 40 μg/mL 5-bromo-4-chloro-indolyl-β-d-galactopyranoside (X-Gal) and verified by blue-white screening and PCR (7) (Table S3).

Purification of Wild-Type and Mutated β-Galactosidase.

To purify β-galactosidase, cultures of Halobacterium sp. NRC-1 transformants (pRK42 and mutant strains) were grown to late-log phase (OD₆₀₀ ∼ 1.0), harvested, resuspended in binding buffer [20 mM phosphate buffer, 2.0 M NaCl, 10% (vol/vol) glycerol, and 30 mM imidazole, pH 7.4], and then sonicated for 15 s at an amplitude of ∼54 μm (Model 50 Sonic Dismembrator; Thermo Fisher Scientific). Cell debris was removed by centrifugation (25,000 × g, 4 °C, 10 min) in an Eppendorf 5417C centrifuge, and the resultant crude extracts were filtered through a 0.2-µm polyethersulfone filter (Thermo Fisher Scientific). The proteins were purified using 1-mL Nickel HisTrap HP columns (GE Healthcare). Each column was equilibrated with binding buffer, and the lysate was loaded onto the column at a flow rate of 1 mL/min. The column was then washed with binding buffer until the absorbance reading was steady. Bound proteins were eluted sequentially with 2 M NaCl and 100 mM phosphate buffer (pH 7.4) supplemented with 40 mM, 80 mM, 100 mM, 200 mM, and 500 mM imidazole. The elutants were collected in five 1-mL fractions for each imidazole concentration.

All column fractions were tested for activity using ONPG (Thermo Fisher Scientific). The three most active fractions were combined for all subsequent work, and protein concentration was estimated by absorption at 280 nm using a Shimadzu UV-VIS 1601 spectrophotometer. To confirm purity, 2-μL aliquots were run on a 10% SDS/PAGE, followed by staining with Coomassie blue (Millipore Sigma).

Purified enzymes were electrophoresed on 6% native polyacrylamide gels and assayed in situ for activity using MUG (3.5 mg/mL in 3.5 M KCl for 30 min at 30 °C) following the manufacturer’s instructions (Sigma-Aldrich). Size was estimated using alcohol dehydrogenase and albumin (Sigma-Aldrich) as molecular weight standards.

Steady-State Kinetic Analysis of Wild-Type and Mutated β-Galactosidase.

Kinetic experiments were performed at temperatures from 0 to 25 °C in 5 °C increments, using a Shimadzu UV-VIS 1601 spectrophotometer with a customized temperature control system designed to prevent condensation at lower temperatures. A solution of 10 µg/mL of enzyme in 2 M KCl and 100 mM PO₄ buffer at pH 6.5 was used for all kinetic reactions. The assay solution was preincubated at the desired temperature for 2 min before the addition of ONPG. Changes in absorption at 420 nm over 2 min and 30 s were recorded. Three different concentrations of ONPG were used: 1, 2.5, and 5 mM). All experiments were run in triplicate and values were averaged. V₀, the Michaelis constant (K_m), and V_max values were determined using Lineweaver–Burk plots with UV Probe ver. 4.23 software (Shimadzu). k_cat values were calculated from V_max and enzyme concentrations. The RSQ function in Microsoft Excel was used to determine R² values of linear regression (Table S4). The LINEST function in Excel was used to determine SEs for K_m, V_max, and k_cat (Table S5).

Homology Modeling.

A structural model of the H. lacusprofundi β-galactosidase was generated by homology modeling using the crystal structure configuration of the T. thermophilus enzyme as a template with Swiss-PDBViewer version 4.1.0. The H. lacusprofundi β-galactosidase model (11) was modified using the mutate tool to generate variants. The probability and s-value for each rotamer were recorded and compared to determine the most likely conformation. Neighbors within 4 Å of the residue side chains were selected and displayed with CPK coloring and rendered in solid 3D. The backbone, side chain, and label were displayed for each residue. The move, zoom, and rotate tools were used to frame the selected residues so that all labels were visible and any steric clashes or hydrogen bonds were observable.