Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060

Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060

Journal of Bioscience and Bioengineering VOL. 121 No. 5, 497e502, 2016 www.elsevier.com/locate/jbiosc Epistasis effects of multiple ancestral-consens...

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Journal of Bioscience and Bioengineering VOL. 121 No. 5, 497e502, 2016 www.elsevier.com/locate/jbiosc

Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060 Yasuhisa Fukuda,1 Asuka Abe,1 Takashi Tamura,1 Takahide Kishimoto,2 Atsushi Sogabe,2 Satoshi Akanuma,3 Shin-ichi Yokobori,4 Akihiko Yamagishi,4 Katsumi Imada,5 and Kenji Inagaki1, * Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan,1 Biochemical Department, Toyobo Co. Ltd., 2-2-8 Dojima Hama, Kita-ku, Osaka 530-8230, Japan,2 Faculty of Human Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359-1192, Japan,3 Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan,4 and Department of Macromolecular Science, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043, Japan5 Received 3 August 2015; accepted 16 September 2015 Available online 20 October 2015

Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (Tm), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60 C (t1/2). Three mutants showed increased stabilities compared to the wildtype protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Glycerol kinase; Thermostabilization; Cellulomonas; Ancestral mutation; Epistasis effect; Diagnostic enzyme]

Glycerol kinase (EC 2.7.1.30; ATP: glycerol 3-phosphotransferase, GK) catalyzes the Mg-ATP-dependent phosphorylation of glycerol to produce glycerol 3-phosphate, which is an important metabolic intermediate for glycolysis (1e4). GK is widely present in all three kingdoms of living organisms, including bacteria, archaea and eukaryotes. GK is a member of the actin-ATPase superfamily, which includes hexokinase, actin and heat shock protein 70 (5). The member proteins of the superfamily share a common bbbababa folding motif, and are known to change their domain conformation greatly upon substrate binding. GK is industrially important and generally used for the clinical determination of the blood triglyceride level together with lipase, glycerol-3-phosphate oxidase and peroxidase (PO) (6). Thermostability and preservative tolerance are crucial properties for the diagnostic application of enzymes. Cellulomonas sp. NT3060 glycerol kinase (CGK) appears to have necessary high preservation tolerance (7). Improving the stability of proteins is still a challenging task. Although various factors responsible for improving the stability of proteins have been proposed to date (8e12), we are still far from a comprehensive understanding of the sequence-structure-stability

* Corresponding author. Tel./fax: þ81 86 251 8299. E-mail address: [email protected] (K. Inagaki).

relationship. Moreover, the effects of an individual factor generally depend on the structural context and therefore the same strategy is not always applicable to attaining greater stability in different proteins. The ancestral design method is an alternative approach for improving the thermostability of proteins that only relies on homologous amino acid sequences of a given protein (13). The method predicted an ancestral amino acid sequence from a multiple amino acid sequence alignment and a phylogenetic tree of homologous proteins. Then, one or a few inferred ancestral residues were introduced into an extant protein as mutation(s). It has been found previously that comparing only a small set of homologous protein sequences is useful for improving the stability of proteins using the ancestral design method (13e16). The consensus approach is also a way to design stable proteins by comparing homologous protein sequences (17). The concept of the consensus approach is that the most frequent amino acid at any position among homologous proteins may contribute to the stability of the protein greater than an amino acid rarely seen in homologous sequences. The predictive worth of the consensus approach has been validated by producing and characterizing full-consensus proteins (18e21). The ancestral design method and the consensus approach frequently result in the same amino acids because consensus residues often originate from ancestral residues. Here we report on the thermostabilization of CGK by changing non-ancestral residues that are rarely seen in the homologous

1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.09.011

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sequence to ancestral residues that are most frequently observed at specific positions in multiple sequence alignments. Some of the resulting mutants showed improved thermostability compared to the wild-type versions of the protein. More interestingly, epistatic effects of individual mutations on the thermostability were observed, meaning that some of the mutations that did not affect the thermostability of CGK when they were introduced individually contributed to further improvements of the stability when introduced in particular combinations. Thus, the best mutant with three amino acid substitutions showed a melting temperature 15 C greater and a half-life 15-fold longer than its wild-type protein. MATERIALS AND METHODS Materials Glycerol-3-phosphate oxidase and Peroxidase were purchased from Toyobo. All other reagents were of analytical grade. Water used was purified with a Milli-Q purification system (Millipore, Billerica, USA). Phylogenetic analysis Phylogenetic tree building and the inferring of ancestral amino acid sequences were performed as reported previously (9). In brief, amino acid sequences of 75 GKs were retrieved from the Genbank database. The amino acid sequences were aligned by Clustal X 1.83 using the default settings and then corrected manually. A maximum likelihood (ML) tree of the dataset was estimated with CODEML in PAML. Using the topology of the ML tree as a guide, we then selected and aligned 17 representative GK sequences for further tree building and the predicting of ancestral bacterial and archaeal sequences. Gblocks (22) with default conditions were used for excluding poorly aligned regions. The remaining 158 sites were used for further ML analysis in order to build an ML tree consisting of 17 GK sequences (Fig. S1). Estimation of ancestral bacterial and archaeal GK sequences was performed using a combination of ancestral sequence predictions by CODEML in PAML (23) and GASP (24). The former model was used to estimate ancestral sequences and the latter model to predict indel positions. Construction of ancestral mutants The mutant Cellulomonas sp. glpK genes were synthesized by a PCR site-directed mutagenesis method. DNA sequences of mutant genes were confirmed by the dideoxy chain termination method using a PRIMS BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Tokyo, Japan) and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Thermostability measurements The wild-type and mutant glpK genes were overexpressed in Escherichia coli KM1 in 1 ml of LB medium supplemented with ampicillin (50 mg/ml) and kanamycin (20 mg/ml). Overexpression was induced by the addition of isopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. After 24 h cultivation at 37 C, the cells were harvested by centrifugation, resuspended in 50 mM potassium phosphate buffer (pH 7.5) containing 0.5 mM EDTA, and then disrupted by sonication. The soluble fractions were regarded as the crude extracts. The crude extracts were incubated at various temperatures for 15 min, or at 60 C for various time periods and then immediately cooled to 0 C. The remaining catalytic activities were estimated. The wild-type and two mutant (T386I/F388Y and A344V/T386I/F388Y) proteins were purified from the soluble fractions of their respective lysates by successively using Q-Sepharose Fast Flow and Phenyl Sepharose 6 Fast Flow (high sub) columns (GE Healthcare, Sweden). The purity of the enzymes was confirmed by native-PAGE. The quantity of the proteins was estimated with a Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Japan) using bovine serum albumin as the standard. The thermal stabilities of the wild-type protein and the two mutants were also assessed using the purified proteins in the same manner as the stability measurement with the crude extracts. Kinetic parameter measurement Enzyme activities were measured by the enzyme-coupling method using glycerol-3-phosphate oxidase and peroxidase (7). The reaction pre-mix contained 0.2 % BSA, 2 mM MgCl2, 0.15 mM EDTA, 0.2 mM 4-aminoantipyrine, 30 mM phenol, 70 units of glycerol-3-phosphate oxidase, 50 units of peroxidase, 3.8 mM ATP and 0.1 M HEPES at pH 7.9. The reaction was initiated by adding 0.3 ml of glycerol and 0.1 ml of an enzyme solution to 2 ml of the pre-mix. The mixture was then incubated at 37 C for 3 min and the catalytic activities of the enzymes were evaluated by measuring the absorbance at 500 nm; this absorbance increase corresponded to the production of quinoneimine dye. The values of the Michaelis constant (Km) for the substrate glycerol and the catalytic constant (kcat) were determined using the results of steady state experiments under various concentrations of glycerol. The kinetic constants were obtained from Lineweaver-Burk plots of the steady-state velocity data.

RESULTS Construction of mutant CGKs A phylogenetic tree consisting of 17 GKs was constructed using the maximum likelihood method

(25). Using this phylogenetic tree, two ancestral GK sequences that were thought to be possessed the common ancestors of bacteria and archaea, were inferred also using the maximal likelihood method. Fig. 1 shows the multiple amino acid sequence alignment of the 17 GKs that were used for tree building aligned with the inferred ancestral sequences. Target positions for amino acid substitution were chosen based on whether they were positions where the same amino acid residue existed in the bacterial and archaeal ancestral sequences but a different amino acid residue was found in the CGK sequence, or places where ancestral amino acid residues were most frequently found at the corresponding positions among the 17 extant GK sequences. Eventually, we constructed seven mutants of CGK. In each of which, a less frequent, non-ancestral amino acid residue was replaced by the most frequent, ancestral residue: Q50E, R202D, L274M, C292A, A344V, T386I and F388Y. The mutants were overexpressed in E. coli KM1 (glpK) and then used for thermostability measurements. Thermostability of the mutant CGKs with a single amino acid substitution Table 1 summarizes the thermal stability of the wild-type and mutant CGKs. The Tm (the apparent halfdenaturation temperature after 15 min incubation) of the wildtype CGK was 53 C. Among the seven mutant CGKs, three (L274M, A344V and T386I) showed higher Tm values than the wild-type CGK, whereas C292A exhibited a lower Tm. We also compared the thermal stabilities of the proteins by measuring the half-denaturation time at 60 C (t1/2). The t1/2 of wild-type CGK was 4 min. The four mutants, L274M, A344V, T386I and F388Y, persisted longer than the wild-type protein. Particularly, F388Y showed the longer t1/2 than did the wild-type protein while its Tm was the same as that of the wild-type. Probably, the temperature dependence of the inactivation rate of F388Y is lower than that of the wild-type CGK. In contrast, the t1/2 value of R202D was shorter than that of the wild-type. Effects of the different combinations of several amino acid substitutions Although some ancestral amino acid residues introduced into CGK improved the proteins’ thermostability, the level of stabilization was not so high. Therefore, we tested combinations of several amino acid substitutions. Because we thought that residues that are close to each other are more likely to affect stability than those that are far apart, we first combined the T386I and F388Y substitutions. The Tm of the resulting T386I/F388Y mutant was found to be 9 C higher than that of the wild-type protein. The t1/2 value of T386I/F388Y was six times longer than that of the wild-type. Thus, the combination of the two substitutions showed a synergistic effect as expected. We further added the Q50E, L274M or A344V substitutions to the T386I/F388Y mutant, producing Q50E/T386I/F388Y, L274M/ T386I/F388Y or A344V/T386I/F388Y. The three mutants were substantially more thermally stable than T386I/F388Y (Table 1). It should also be noted that the single substitution of Q50E did not affect thermostability. The observation exemplified the idea that the effects of individual amino acid substitutions on the thermal stability of proteins are not always additive and that the effect of a mutation may be affected by its surroundings. The mutant with a quadruple substitution (Q50E/L274M/T386I/F388Y) is less stable than the mutant with the triple substitution (Q50E/T386I/F388Y). Thus, it is also possible that the presence of one mutation negatively impact the stabilizing effect of other mutations (see Discussion for more details). Thermostabilities of purified proteins The wild-type protein, T386I/F388Y and the most thermally stable mutant, A344V/T386I/F388Y, were characterized further after being purified to homogeneity by column chromatography. The Tm values of the purified T386I/F388Y and A344V/T386I/F388Y

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FIG. 1. Multiple alignment of glycerol kinase from selected organisms. The bacterial ancestor and the archaeal ancestor are ancestral sequences reconstructed using the maximum likelihood method.

TABLE 1. Thermal stabilities of the wild-type and mutants of Cellulomonas sp. NT3060 glycerol kinase analyzed using crude extracts. Tma ( C)

Protein Wild-type Q50E R202D L274M C292A A344V T386I F388Y T386I/F388Y Q50E/T386I/F388Y L274M/T386I/F388Y A344V/T386I/F388Y Q50E/L274M/T386I/F388Y a

53 53 53 58 52 55 56 53 62 69 64 68 68

t1/2b (min) c

(0) (0) (0) (þ5) (1) (þ2) (þ3) (0) (þ9) (þ16) (þ11) (þ15) (þ15)

4 4 3 9 6 4 7 8 24 32 29 59 20

(1.0)d (1.0) (0.75) (2.3) (1.5) (1.0) (1.8) (2.0) (6.0) (8.0) (7.3) (15) (5.0)

Tm, the apparent half-denaturation temperature when treated for 15 min. t1/2, the apparent half-denaturation time when treated at 60 C. c The figures in parentheses indicate the difference from the wild-type enzyme. d The figures in parentheses indicate the difference from the relative values normalized to the value for the wild-type. b

mutants were 7 C and 18 C higher than that of the wild-type protein, respectively. The t1/2 values of T386I/F388Y and A344V/ T386I/F388Y were 4.3 and 28 times longer than the wild-type proteins, respectively (Table 2). These results are almost parallel with those obtained using crude extracts of E. coli expressing the respective proteins. Catalytic properties The kinetic parameters of the wild-type protein, and mutants were obtained from steady-state experimental data (Table 3). The Km values of the purified T386I/ F388Y and A344V/T386I/F388Y mutant proteins for glycerol were about 1.3 and 2.3 fold greater than that of the wild-type protein, respectively. In addition, the kcat values of both mutants were about 50% of that of the wild-type. Therefore, the mutants showed decreased kcat/Km values compared with the wild-type proteins. Thus, the thermal stabilities of T386I/F388Y and A344V/ T386I/F388Y were improved at the cost of catalytic efficiency. DISCUSSION The ancestral design method is a semi-rational way for a designing thermally stable proteins, and only relies on amino acid

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sequences of homologous enzymes (8). The concept is that, given their (hyper)thermophilic ancestry (26), ancestral residues can be expected to contribute to the thermostability of a protein to a much greater extent than is possible with non-ancestral residues. The validity of the ancestral design method has been tested previously by constructing mutant enzymes in which one or a small number of non-ancestral, original residue(s) are replaced with inferred ancestral amino acid(s) (9,17,27,28). The resulting mutants show a pronounced trend toward enhanced thermal stability when compared to the wild-type protein. The consensus approach is also a way of improving the thermal stability of a protein using a multiple sequence alignment of homologous proteins (29). The theoretical basis of the consensus approach is that the most frequent amino acid residue at a given position in a multiple sequence alignment of homologous proteins contributes to the thermostability of the protein more than other less frequent amino acids. However, it is never certain that all stabilizing consensus residues can be always identified correctly. Its success often relies on obtaining a phylogenetically unbiased sampling of the sequence set included in the multiple sequence alignment (30). Conversely, the ancestral design method can, at least partially, make up for erroneous predictions of consensus residues that could be caused by selection bias during sequence collection (31). In the current study, we replaced non-ancestral, less frequent amino acid residues in CGK by the inferred ancestral residues that are the most frequent at respective positions in the multiple sequence alignment, thus producing seven mutants with a single amino acid substitution. Thermal stability measurements showed that three mutants had improved thermal stability, another three were neutral, and only one was less stable than wild-type CGK. Even for the best mutant, L274M, its Tm was only 5 C higher and its t1/2 was only 2.3 fold longer than those of the wild-type. We considered that, even if the effects of individual mutations on the stability are small, presence of multiple mutations in particular combinations may enhance the stabilizing effects of the individual mutations. We thought that amino acid substitutions at positions that are close each other are more likely to show synergistic effects on stability, than those that are farther apart. Of the seven mutations that were individually introduced into CGK, only T386I and F388Y are located within 5 Å of each other. We therefore constructed the T386I/F388Y mutant. Thermal stability measurements revealed that T386I/F388Y was thermally much more stable than

TABLE 2. Thermal stabilities of the purified wild-type and mutants of Cellulomonas sp. NT3060 glycerol kinase. Tma ( C)

Protein Wild-type T386I/F388Y A344V/T386I/F388Y

t1/2b (min) c

53 61 71

(0) (þ8) (þ18)

(1.0)d (4.3) (28)

4 17 112

a

Tm, the apparent half-denaturation temperature when treated for 15 min. t1/2, the apparent half-denaturation time when treated at 60 C. The figures in parentheses indicate the difference from the wild-type enzyme. d The figures in parentheses indicate the difference from the relative values normalized to the value for the wild-type. b c

TABLE 3. Kinetic parameters of the wild-type and mutants of Cellulomonas sp. NT3060 glycerol kinase. Protein Wild-type T386I/F388Y A344V/T386I/F388Y

Km for glycerol (mM)a

kcat (s1)a

kcat/Km (mM1 s1)

36 49 87

4900 2600 2500

136 53 29

a The kinetic parameters of the wild-type and mutant enzymes were determined by fitting the results of steady-state kinetic experiments to the MichaeliseMenten equation.

expected, indicating that the effect of a mutation on protein stability can be heavily affected by its surroundings. Similarly, the addition of the Q50E substitution to T386I/F388Y further improved the thermostability even though the effect of the single substitution of Q50E was neutral. The A344V mutant showed only 2 C higher than that of the wild-type protein and its t1/2 value was the same as that of the wild-type. Nevertheless, the addition of A344V mutation to T386I/F388Y further improved the Tm value by 6 C and the t1/2 values by 2.5 fold. These results exemplified the epistatic effects of the amino acid substitutions on the thermostability of the protein, meaning that an amino acid substitution that does not or slightly improves thermostability can substantially stabilize the protein if it is introduced in combination with other mutations. Some of the epistatic effects of the individual mutations on the thermostability can be explained by mapping the mutation sites on the CGK structure available in PDB (2d4w) (Fig. 2). CGK is composed of two domains, domain-1 and domain-2, and forms a homodimer. Domain-2 is responsible for the dimer’s formation. Gln50 and Arg202 are located in domain-1 and Leu274, Cys292, Ala344, Thr386 and Phe388 are in domain-2. Ala344 is present in the vicinity of the dimer interface but does not directly interact with the residues of another subunit. Rather, the aliphatic side chain forms a hydrophobic core together with Phe346, Leu366, Leu369, Val373 and Ile378 of the same subunit. Therefore, we consider that the substitution of Ala344 by Val enhanced the thermostability through the improvement of the hydrophobicity and packing density of the interior core. Cys292 exists on the interface between domain-1 and domain-2. It has been pointed out that a mutation at the interface between domains can stabilize a multi-domain protein through the rearrangement of the domains (32). It could be possible that the longer t1/2 observed for the C292A mutation was caused by a similar structural mechanism in CGK. All single mutation sites that increase the thermostability (L274M, A344V and T386I) are present in domain-2. This observation implies that, in the wild-type protein, domain-2 is less thermally stable than domain-1 and the thermal denaturation probably starts from domain-2. If this is the case, the stabilizing effect of Q50E in domain-1 may be hidden by the denaturation effect of domain-2 but emerges with the stabilization of domain-2 by the double mutation of T386I/F388Y. In wild-type CGK, the side chain oxygen of Gln50 interacts with the main-chain NH group of Asp-35 and the side chain NH2 group is exposed on the molecular surface. The mutation to glutamate replaces the side chain NH2 group with a carboxyl oxygen, and this substitution may allow additional hydrophilic contact of its side chain with the main-chain NH group of Asn-47. As a result, the glutamate side chain may bridge the Asp-35 and Asn-47, which are both located at the proximal ends of the bturn-b motif in domain-1, and thereby stabilize domain-1. However, the substitution of Gln50 by Glu led to the increased Tm and longer t1/2 of the protein only when the domain-2 was also stabilized by the double mutation of T386I/F388Y, as mentioned above. All single mutations that raised thermostability were substitutions to larger hydrophobic residues, and may have strengthened the hydrophobic interaction in the core of domain-2. This fact suggests that the major cause of the instability of domain-2 is the loose packing of the hydrophobic core. The mutation of F388Y likely does not change the hydrophobic core’s structure, thus the single mutation had no effect on thermostability. However, the F388Y mutation may contribute to an increase in the hydrogen bonding network with Glu384 and Asn341. This contribution may appear with the stabilization of the hydrophobic core by the T386I mutation. We can interpret the effects of the mutation(s) on thermal stability by mapping the sites on a 3D-structure, but it is very difficult to predict the actual effects of the mutation(s) on the thermal stability of an enzyme, and much more so to design a thermostabile

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FIG. 2. Mapping of the seven mutation sites on the crystal structure of glycerol kinase from Cellulomonas sp. NT3060 (CGK). (A) Ribbon representation of the CGK dimer. One subunit is colored in blue and the other in cyan (domain-1) and gray (domain-2). The residues mutated into the ancestral residues are labeled and shown by the ball-model in magenta. (B) The model viewed from the right side of panel A. This image was drawn using PyMOL (http://www.pymol.org). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

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