Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432 Horinouchi, Hachioji, Tokyo 192-0392, Japan
1 To whom correspondence should be addressed. e-mail: yamagish{at}ls.toyaku.ac.jp
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Abstract |
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Keywords: dimeric enzyme/hydrophobic core/isopropylmalate dehydrogenase/subunit interaction/thermostability
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Introduction |
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3-Isopropylmalate dehydrogenase (IPMDH) is a homodimeric enzyme of the leucine biosynthetic pathway. Genes encoding this enzyme have been cloned and sequenced from a variety of sources. The three-dimensional structure of the IPMDH from an extreme thermophile, Thermus thermophilus (TtIPMDH), showed it to be composed of two identical subunits. The subunitsubunit interaction of IPMDH is maintained by the four-helix bundle formed by the two helices of each subunit and the hydrophobic core of the bundle composed of some hydrophobic residues (Imada et al., 1991). The hydrophobic region at the subunit interface was highly conserved between the thermophile and mesophile IPMDHs from Escherichia coli (EcIPMDH) and Bacillus subtilis (BsIPMDH). However, Leu246 and Val249 in T.thermophilus are Glu and Met, respectively, in the corresponding mesophilic enzymes. When Glu256 of EcIPMDH and Met259 of EcIPMDH in the hydrophobic core were substituted with Leu and Val, respectively, which are the corresponding residues in TtIPMDH, thermostability increased (Kirino et al., 1994
). Mutated B.subtilis IPMDH (Glu253Leu or Met256Val) also displayed elevated thermal stability compared with B.subtilis IPMDH without these mutations (Akanuma et al., 1999
). These results were interpreted as meaning that hydrophobic interactions at the subunit interface are critically important for the thermostability of dimeric enzymes. In this study, we have examined the role of Met256 in the hydrophobic core of the subunit interface of BsIPMDH by mutating this position and analyzing the thermal stability of the resulting proteins.
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Materials and methods |
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3-Isopropylmalate and NAD were purchased from Wako Pure Chemicals and Orient Yeast Ltd, respectively (Tokyo, Japan). All other reagents were of analytical grade.
Construction of mutated enzymes.
Site-directed mutagenesis was carried out by PCR according to the methods of Picard et al. (Picard et al., 1994). Plasmid pET21c, which harbors the gene for BsIPMDH, was used as the template. The sequences of the flanking primers used for mutagenic PCR were 5'-TAATACGACTCACTATAGGG-3' and 5'-CTAGTTATTGCTCAGCGGT-3'. The primers used for mutagenesis were 5'-TAAGCGATGAAGCGTCCSY TCTTACAGGCTCGCTCGG-3' for substitution of Met256 to Ala, Val or Phe, and 5'-TAAGCGATGAAGCGTCCM TCCTTACAGGCTCGCTCGG-3' for substitution of Met 256 to Ile or Leu. Mutations were confirmed by sequence analysis (Sanger et al., 1977
).
Enzyme purification
Expression and purification of the wild-type and mutated IPMDHs were carried out as described previously (Hayashi-Iwasaki and Oshima, 2000). Wild-type and mutant IPMDHs were overexpressed in E.coli BL21 (
leuB) cells harboring the recombinant plasmid constructed using a pET21c expression vector. Cells were harvested and disrupted by sonication and the cell-free extract was clarified by centrifugation. The enzyme was purified by standard column chromatography (Hayashi-Iwasaki and Oshima, 2000
), using Hiprep Q (Amersham Pharmacia Biotech, Uppsala, Sweden), Phenyl Toyopearl (Toso, Tokyo, Japan) and Resource Q (Amersham Pharmacia Biotech) columns.
Enzyme assay
The enzyme activity was measured in 100 mM potassium phosphate buffer (pH 7.6), containing 1 M KCl, 0.2 mM MnCl2, 0.8 mM D,L-isopropylmalate and 0.8 mM NAD, by monitoring the absorbance at 340 nm, using a Beckman DU7400 spectrophotometer, at 40°C, according to the method described previously (Yamada et al., 1990).
Measurement of thermal stability against irreversible inactivation
The residual activity of enzyme after heat treatment was estimated according to the previously described method (Kotsuka et al., 1996). The enzyme was diluted to 0.4 mg/ml with 20 mM potassium phosphate (pH 7.6), containing 0.5 mM EDTA, and incubated at various temperatures for 10 min. After the heat treatment, the samples were immediately cooled on ice for 10 min and centrifuged at 21 000 g for 10 min. The residual activity of the supernatant was measured at 40°C. Thermal denaturation was monitored by circular dichroism (CD) at 222 nm with a Jasco J-720C spectropolarimeter (Iwasaki et al., 1996
). The enzyme was dissolved (0.2 mg/ml) in 20 mM potassium phosphate (pH 7.6). The temperature of the enzyme solution was controlled with a circulating bath and increased with a programmable temperature controller (Neslab; Thermo Electron Co., Waltham, MA) at a scan rate of 1.0°C/min. The temperature of the sample was monitored with a thermocouple.
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Results |
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To examine the relationship between hydrophobicity of residue 256 and the thermostability of BsIPMDH, we prepared the mutant enzyme by site-directed mutagenesis in which Met256 was substituted with Ala, Val, Leu, Ile or Phe. Mutated forms of BsIPMDH were overexpressed in E.coli BL21 (leuB) cells and purified to homogeneity.
The thermal stability of the purified mutated BsIPMDHs were estimated by measuring the residual activities at 40°C after heat treatment at various temperatures for 10 min. The thermal inactivation processes of the wild-type and mutant BsIPMDHs were irreversible under the conditions used. Figure 1 shows the residual activities after heat treatment. The specific activity of all non-heat-treated mutant BsIPMDHs did not differ from that of the wild-type BsIPMDH by >5% (data not shown). The Th (temperature at which half of the protein is denatured) of BsIPMDH-M256V was 2°C higher than that of wild-type BsIPMDH, but was unchanged for BsIPMDH-M256L and BsIPMDH-M256I despite the substitution of more hydrophobic residues. Furthermore, the Th of BsIPMDH-M256F was 4.5°C lower than that of the wild-type BsIPMDH. In contrast, BsIPMDH-M256A had a Th 6.0°C higher than that of wild-type BsIPMDH. These results indicate that the effect of hydrophobicity at position 256 on the thermal stability is contrary to expectations.
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If the hydrophobicity of the amino acid residue at position 256 is the principal factor governing thermostability, we would predict that IPMDHs with a more hydrophobic residue (BsIPMDH-M256I, -M256L and -M256F) should have an elevated Tm whereas the mutated form with a less hydrophobic residue (BsIPMDH-M256A) would have a lower Tm. Thus, our experimental findings are contrary to predictions. An increase in thermostability was detected for mutated proteins where a smaller amino acid residue, such as Ala or Val, was substituted at position 256. We examined the relationship between Th and Tm against the van der Waals volume of the amino acid residue 256 (Figure 3). The Th or Tm decreased depending on the increase in the van der Waals volume of the amino acid residue at position 256. The Tm of the M256F variant deviated from the Th, but the Tm of the second phase of the denaturation curve was similar to the Th.
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Figure 4 shows the side view of the dimer interface which is composed of hydrophobic residues from four helices; two helices from each subunit. Figure 5 shows the bottom view of the four-helix bundle at the interface. In TtIPMDH, Val224 residues from two subunits are located at the center of the four-helix bundle. Val224 and Val224' are sandwiched between Val249 and Val249'. Although Val249 and Val249' are located close to the outer surface of the enzyme, they are almost buried, having a solute-accessible surface area of only 3.1%. The corresponding residues in EcIPMDH have a similar conformation in which Ile234 and Ile234' (Ile231 and Ile231' of BsIPMDH) are surrounded by Met259 and Met259' (Met256 and Met256' of BsIPMDH). The Met259 residue in EcIPMDH is still buried and has a solvent-accessible area of 3.1%. In this study, proteins with a smaller amino acid residue at position 256 of BsIPMDH (EcIPMDH-M259) showed increased thermal stability against irreversible inactivation, indicating an inverse relationship between the enzymes thermostability and the van der Waals volume of the amino acid residue.
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Discussion |
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In this study using BsIPMDH, we found that the single-mutation M256V increased thermostability over the wild-type enzyme, supporting the previous interpretation. However, as shown in Figure 3, hydrophobicity of the amino acid residue at position 256 was inversely correlated to the thermal stability against irreversible inactivation of BsIPMDH. A mutated enzyme carrying a smaller amino acid residue at position 256 showed higher thermostability.
Denaturation of wild-type BsIPMDH was irreversible. Accordingly, the denaturation curve represents the denatured form accumulated during the gradual increase in temperature during the CD measurement. However, residual activity was estimated after the heat treatment for 10 min [a 10 min denaturation has been used for the estimation of the thermal stability of IPMDHs for the other organisms (Yamada et al., 1990; Numata et al., 1995
; Yoda et al., 1995
; Wallon et al., 1997
b; Tamakoshi et al., 2001
)]. The residual activity is expected to be directly proportional to the fraction of native enzyme after the 10 min heat treatment. Thus, the Th estimated by residual activity does not necessarily coincide with the Tm estimated by CD measurement. However, the temperatures estimated by these two methods were similar for each mutated protein (Figure 3).
A two-phase denaturation curve was observed in the CD measurement of BsIPMDH-M256F (Figure 2). It took 10 min to increase the temperature of the protein solution from 30 to 40°C. Thus, the first phase of the denaturation process, monitored by CD, was expected to be complete after treatment at 40°C for 10 min. However, when the residual activity of BsIPMDH-M256F after the 10 min heat treatment was estimated (Figure 1), we observed only a 10% decrease in activity. Therefore, the first phase of the BsIPMDH-M256F denaturation curve measured by CD appears to be reversible or at least unrelated to the loss of activity. Though detailed analysis may be necessary to elucidate the characteristics of each phase of the denaturation curve, the Tm of the second phase was rather closer to the Th estimated by the residual activity in BsIPMDH-M256F.
Figure 4 shows the side view of the helix-bundle structures of TtIPMDH and EcIPMDH observed in the plane of the subunitsubunit interface. From this perspective, the distance between the two helices of the different subunits is even from top to bottom in EcIPMDH while the distance is shorter at the bottom in TtIPMDH. The corresponding amino acid residues at the bottom of the helices are closer in TtIPMDH than EcIPMDH: the distance between the carbons of Val249 and Val224' is 7.4 Å in TtIPMDH, while that of Met259 and Ile234' is 7.7 Å in EcIPMDH.
In the structure of TtIPMDH, the distance between the carbon atom of V249 and the
carbon atom of V224' is 4.0 Å (Figure 5A). Val249 is also surrounded and sandwiched by Met221' and Arg225' residues from the top and from the bottom of Val249, respectively (not shown in Figure 5). In the structure of EcIPMDH, Met259 and Ile234' are also in close contact: the distance between the
carbon of Met259 and the
2 carbon of Ile234' is 4.0 Å. The Met259 residue is also surrounded and sandwiched by Met231' and Lys235' residues. Although still buried, the sulfur atom and
carbon of Met259 project outwards. Thus, the total volume occupied by the amino acid residues in this region in EcIPMDH is larger than that in TtIPMDH. The combination of these residues appears to determine the distance between the two helices in these proteins. Substitution of residues with a larger van der Waals volume may have introduced stress in the conformation of side chains at the subunit interface. Alternatively for EcIPMDH, the bulky residues in this region may have increased the distance between the helices, thereby affecting the interaction immediately above these residues.
Table I shows residues corresponding to I231 and M256 of BsIPMDH in IPMDHs from other microorganisms. Combinations of the corresponding residues are different in different organisms. Although different, none of the combinations of two residues have large volumes except for BsIPMDH and EcIPMDH. Interestingly, for the ultrathermophile Thermotoga maritima, there are hydrophobic residues at the two corresponding sites (I231 and A256). This combination is the same as that of BsIPMDH-M256A that showed the highest thermal stability among the BsIPMDH-M256 mutants tested.
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Glaser et al. determined the residueresidue preferences in intermolecular interactions based on a database of numerous co-crystallized proteinprotein interfaces (Glaser et al., 2001). Their results revealed that the highest residueresidue preferences at the interface were for interactions between pairs of large hydrophobic residues, and the lowest preferences for interactions between pairs of small residues. Experimental studies by Vallone et al. have indicated that there is a correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area (Vallone et al., 1998
). Thus, the hydrophobicity at the subunit interface is expected to be the main driving force for proteinprotein interactions.
Nevertheless, hydrophobic packing, rather than hydrophobicity alone, at the hydrophobic core of the protein interior, is essential for the stability of the native conformation of proteins. Lim and Sauer suggested that the total volume and steric interaction of residues in the hydrophobic core is the limiting factor for protein stability based on their experiment involving random mutagenesis (Lim and Sauer, 1989). In this study, we found that the volume of an amino acid residue in the hydrophobic core at the subunit interface is also important for the stability of a dimeric protein. In conclusion, hydrophobic packing, rather than hydrophobicity at the interface, appears to be a crucial factor in proteinprotein interaction.
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Acknowledgements |
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References |
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Received September 3, 2002; revised June 13, 2003; accepted June 20, 2003.