1 Asahi Kasei Corporation, Shizuoka 410-2321, 2 Molecular Gene Technics, Genetic Resource Technology, Kyushu University, Fukuoka 812-8581 and 3 Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan
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Abstract |
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Keywords: Flavobacterium meningosepticum/glycerol kinase/random mutagenesis/thermostable mutant enzyme
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Introduction |
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In a previous paper, we reported the purification of GK from F.meningosepticum (FGK) and characterized it (Sakasegawa et al., 1998). Although F.meningosepticum is a typical mesophile, the thermostability of FGK is higher than that of GK from a moderate thermophile, B.stearothermophilus (Comer et al., 1979
) and comparable to that of GK from the extreme thermophile, T.flavus (Huang et al., 1998
). The FGK has been cloned and abundantly produced from the recombinant E.coli cells. In addition, FGK is stable at 37°C for a long period of time. Such a high stability of FGK under various conditions is advantageous for easy preparation of the pure enzyme, for elucidation of the relationship between the structure and function, and for the diagnostic analysis of triglycerides and lipids in the serum.
Determination of the structural features contributing to the thermostability of many proteins has been approached both through comparison of the amino acid sequences and the crystal structures of homologous mesophilic and thermophilic proteins and through protein engineering. As a result, many structural features have been suggested and the stability of the thermophilic proteins can depend on the combination of these factors (for example, Matthews, 1993; Tanner et al., 1996
; Auerbach et al., 1998
; Tahirov et al., 1998
; Britton et al., 1999
; Hashimoto et al., 1999
; Natesh et al., 1999
; Vieille and Zeikus , 2001
).
In this study, to obtain more thermostable FGK and further information about the enhancement of thermostability, the enzyme was investigated based on the tertiary structure of E.coli GK (Hurley et al., 1993) by the random mutagenesis that focused on the subunit interface region.
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Materials and methods |
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Glycerol-3-phosphate oxidase was purchased from Asahi Kasei Corporation (Tokyo, Japan). ATP, ampicillin and the peroxidase were obtained from Sigma Chemical (St Louis, MO), and the brain heart infusion (BHI) was from Difco Laboratories, (Detroit, Mich.). Plasmid pUC118, restriction endonucleases, DNA modifying enzymes and all other genetic engineering kits were purchased from Takara (Kyoto, Japan), while N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS) was from Dojindo (Kumamoto, Japan). DEAE-Sepharose Fast Flow, Phenyl-Sepharose Fast Flow, Q-Sepharose Fast Flow and Sephadex G-25 were obtained from Pharmacia, Sweden. All other chemicals were analytical grade products from Nacalai Tesque (Kyoto, Japan).
Determination of enzymatic activity
The enzyme assay was carried out as previously described (Sakasegawa et al., 1998). The standard reaction mixture contained 50 mM TrisHCl buffer (pH 8.0), 1 mM glycerol, 4 mM ATP, 4 mM MgCl2, 10 units of glycerol-3-phosphate oxidase, 4.5 units of peroxidase, 1.5 mM 4-aminoantipyrine, 1.5 mM DAOS and 20 µl of enzyme in a final volume of 1.02 ml. The reaction mixture was incubated at 37°C in a cuvette with a 1.0-cm light path and the increase in absorbance at 600 nm [
M (molar absorption coefficient) = 17.5/mM/cm] was spectrophotometrically measured. One unit (U) of enzyme was defined as the amount of enzyme catalyzing the formation of 1 µmol of glycerol-3-phosphate per minute at 37°C. Specific activity was expressed as U/mg protein. Protein was measured with a Bio-Rad protein assay kit using bovine serum albumin as the standard protein.
The apparent kinetic parameters, Km and kcat, for glycerol and ATP were estimated from the LineweaverBurk plots. The concentrations of ATP and glycerol used for the kinetic parameters determination were in the ranges of 02.0 and 00.05 mM, respectively.
Random mutagenesis
Random mutagenesis of the FGK was carried out using a Mutan-K kit according to the method of Kunkel (Kunkel, 1985). A 2.4 kb EcoRISphI fragment containing the FGK gene was excised from an expression plasmid of the wild-type FGK that was previously constructed (Sakasegawa et al., 1998
). After filling up of the EcoRI site by use of a DNA Blunting kit, the fragment was subcloned into the SmaISphI site downstream of the lac promoter of pUC118. This recombinant plasmid was incorporated into E.coli CJ236 and the cell was transformed to yield the uracil-containing single-strand DNA. Mix primers for mutagenesis (5'-CAT GAC GCG GCA NNN GTA NNN NNN CTT GCG NNN NNN GTT AAG GAT AAT-3'; N indicates a mixture of A, G, C and T) were synthesized using an Applied Biosystems Model 394 DNA synthesizer and were annealed to the single-strand DNAs. The second DNAs were synthesized using the T4 DNA polymerase. After ligation with E.coli ligase, the E.coli BMH71-18mutS was transformed with the double-stranded heteroduplex DNA. The introduced base substitution was confirmed by DNA sequencing.
Isolation of the mutant exhibiting higher thermostable GK
Transformants were spread onto 3.7% BHI agar plates containing 50 mg/l ampicillin and 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). After the plates were incubated at 30°C overnight, each colony was picked up and grown at 30°C in 3.7% BHI liquid medium containing 50 mg/l ampicillin and 1 mM IPTG. The cells were harvested, suspended in 20 mM potassium phosphate buffer (pH 7.0) and disrupted by sonication. The supernatant obtained by centrifugation (8000 g, 20 min) was heated at 73.5°C for 30 min. The heat stability was evaluated by comparison with the remaining activities after heat treatment.
Site-directed mutagenesis
Four additional mutants were obtained according to the method described by Kunkel et al. (Kunkel et al., 1987). Sequences of the primers used for the mutagenesis were 5'-CAT GAC GCG GCA GAC GTC AGT ACT CTT GCG GGC AGC GTT AAG GAT AAT-3', 5'-CAT GAC GCG GCA GAA GTC GAC ACT CTT GCG GGC AGC GTT AAG GAT AAT-3', 5'-CAT GAC GCG GCA GAA GTA AGT GCA CTT GCG GGC AGC GTT AAG GAT AAT-3' and 5'-CAT GAC GCG GCA GAA GTA AGT ACT CTT GCG GGC AAG GTT AAG GAT AAT-3' for Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys, respectively.
Expression and purification of the mutants
The mutated FGKs were expressed in E.coli DH5. All purification procedures were done at room temperature, and 50 mM TrisHCl buffer (pH 8.0) was used as the standard buffer throughout the purification. The recombinant strain producing each of the mutated FGKs was cultured in a 3.7% BHI liquid medium with 50 mg/l ampicillin and 1 mM IPTG at 37°C for 17 h. The cells were suspended in the buffer and disrupted by sonication. The cell debris was removed by centrifugation (8000 g, 10 min), followed by heat treatment at 65°C for 30 min. After centrifugation (8000 g, 15 min), the supernatant solution was put on a DEAE-Sepharose Fast Flow column equilibrated with the buffer. The enzyme was eluted with a linear gradient of 00.5 M KCl. The fractions with activity were collected, solid (NH4)2SO4 was added (20%), and the solution was put on a Phenyl-Sepharose Fast Flow column equilibrated with the buffer containing 20% (NH4)2SO4. The enzyme was eluted with a linear gradient of the concentration from 20 to 0% (NH4)2SO4. The desalted enzyme solution was then put on a Q-Sepharose Fast Flow column equilibrated with the buffer. The bound enzyme was eluted with a linear gradient of 00.5 M KCl. The active fractions were collected, concentrated and desalted by passage through a Sephadex G-25 column.
Heat inactivation
The enzyme solution (25 µg/ml in 50 mM TrisHCl buffer pH 8.0) was incubated at various temperatures for certain periods of time, followed by rapid cooling. The remaining activity after the treatment was assayed. The kd [first-order rate constant (s1)] of heat inactivation was calculated as described elsewhere (Suzuki et al., 1980).
Structural model construction of FGK and FGK2615
The FGK sequence has shown a 60% homology with that of E.coli GK (Sakasegawa et al., 1998). The crystal structure of the complex of E.coli GK with the unphosphorylated form of IIIGlc (an allosteric inhibitor of the phosphotransferase system) has already been determined by Hurley et al. (Hurley et al., 1993
). The E.coli GK coordinates (1glb) were obtained from the Brookhaven Protein Data Bank as a template. The model was constructed using the InsightII/Homology software package available from Molecular Simulation Inc., San Diego, CA. Side-chain substitutions were automatically done by the InsightII program. After the substitutions, structure relaxation was performed with the steepest descent method under the conditions restricting the relative position of C
. The optimization was done using the conjugate gradient method until the maximum derivative was less than 0.01 kcal/Å. All geometry optimization operations were performed using the consistent valence force field in the program Discover available from Molecular Simulation Inc.
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Results |
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The region from Lys320 to Val335 was chosen as the target for the random mutagenesis to increase the thermal stability. In this region, we chose especially the residues at the positions of Glu327, Ser329, Thr330, Gly333 and Ser334 for the random mutagenesis to introduce the further increase of hydrophobicity in this region and to elevate thermostability of the enzyme (Figure 1). Thermal stability of the enzyme in the crude extract obtained from each of 2000 clones was examined. As a result, we found one colony producing a higher thermostable GK than the FGK. The remaining activity of the crude mutant enzyme (FGK2615) was 74% after heat treatment at 73.5°C for 30 min, but that of FGK was 52% under the same conditions. The thermostability of the other transformants was similar to or lower than that of the FGK. A recombinant plasmid was extracted from these clone cells and the DNA sequence of the insert fragments was determined. The changes in eight bases (5'-CAT GAC GCG GCA GAT GTA GAT GCA CTT GCG GGC AAG GTT AAG GAT AAT-3'; the italic letters indicate the positions of nucleotide mutations) were found in the gene which resulted in the replacement of four amino acid residues in the sequence of FGK2615 (Figure 1
).
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FGK2615 was purified approximately 200-fold with a high yield of 75% to show homogeneity on SDSPAGE. The thermostability of FGK2615 was compared with that of the parent enzyme (Figure 2). The FGK2615 clearly exhibited a higher thermostability than the parent enzyme. The optimum pH, specific activity, temperature depending on the activity and the effect of pH on the stability for the mutant enzyme were similar to those of the parent enzyme. The Km values of FGK2615 for glycerol and ATP were determined to be 3.6 and 521 µM, respectively. This shows that the Km for the ATP of FGK2615 is approximately twice that of FGK (249 µM) but a substantial change in Km for glycerol was not observed between FGK (3.5 µM) and FGK2615. No significant alternations in the kcat values for the ATP of FGK and FGK2615 were observed (Table I
).
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FGK and FGK2615 followed the first-order kinetics of heat inactivation at pH 8.0 and gave linear Arrhenius plots of the kd values (Figure 3). The T50 values (the temperature at which the enzyme activity is inactivated by 50% for a 30-min incubation) were calculated to be 69.0 and 72.1°C, respectively (Table I
).
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Four additional mutants were constructed by site-directed mutagenesis, i.e. E327D (Glu327 to Asp), S329D (Ser329 to Asp), T330A (Thr330 to Ala) and S334K (Ser334 to Lys). Of the four mutants, the three mutants of E327D, T330A and S334K exhibited a similar thermostability to the parent enzyme (Figure 3). On the other hand, the S329D showed a substantially increased thermostability; the T50 (72.6°C) for the S329D was compatible with that (72.1°C) for the FGK2615 (Table I
). In addition, the S329D showed a Km (586 µM) value for ATP which was also similar to that (521 µM) of the FGK2615.
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Discussion |
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In recent years, based on three-dimensional structures, many molecular and structural characteristics of the highly thermostable enzyme have been analyzed by pairwise comparisons with homologous proteins which have different thermal stabilities (for example, Tanner et al., 1996; Auerbach et al., 1998
; Sanz-Aparicio et al. 1998
; Tahirov et al., 1998
; Britton et al., 1999
; Hashimoto et al., 1999
; Natesh et al., 1999
). As a result, several mechanisms and factors have been found to be responsible for the higher thermostability, i.e. hydrophobic interactions, ion-pairs, hydrogen bonds, helix capping, solvent accessible surface areas, etc. (Matthews, 1993
; Vieille and Zeikus, 2001
). Among them, the large contribution of hydrophobic interactions in the subunit interface to the stability has been demonstrated with some thermophilic enzymes (Biro et al., 1990
; Kirino et al., 1994
; Erduran and Kocabiyik, 1998
). Escherichia coli GK is known to exist in the equilibrium state between the functional dimer and tetramer subunit structures for the physiological concentration (de Riel and Paulus, 1978
). In the GK structure, three types of intersubunit interactions are observed (Hurley et al., 1993
). Although the entire hydrophobic profile (Kyte and Doolittle, 1982
) of FGK is very similar to that of the E.coli GK, the FGK exhibits higher hydrophobicity only in part of the subunitsubunit interface region than that of E.coli GK; the subunitsubunit interface region is Lys320 to Ala332 for FGK, and the region with higher hydrophobicity in FGK compared to E.coli GK is Asp324 to Val335 (Figures 1 and 4
). For this point, the FGK is clearly different from the E.coli GK (Sakasegawa et al., 1998
). We postulated that this region may be responsible for the higher thermostability of the FGK and then carried out the random mutagenesis in the region of FGK as the strategy for increasing the thermostability. As a result, one clone, which produces a mutant enzyme with a higher thermostability than the parent FGK, was obtained and four amino acid substitutions, Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys, were identified in the mutant enzyme (Figure 1
). Unlike our prediction, the replacement in the mutant enzyme did not result in a hydrophobicity increase in the interface region. We tried the construction of additional enzyme mutants in order to determine which substitution among the four amino acids was exclusively responsible for the increased thermostability. The result clearly showed that only the mutation of Ser329 to Asp and not the other three mutations is responsible for the increased thermostability of FGK2615 and S329D (Figure 3
). This indicates that the change in the side chain of Ser329 to Asp mainly produces the change of its nature.
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Notes |
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Acknowledgments |
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References |
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Received January 15, 2001; revised June 20, 2001; accepted June 23, 2001.