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Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties.

Byun JS, Rhee JK, Kim ND, Yoon J, Kim DU, Koh E, Oh JW, Cho HS - BMC Struct. Biol. (2007)

Bottom Line: The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other alpha/beta hydrolases.In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1.Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul, Korea. lovemilk99@yonsei.ac.kr <lovemilk99@yonsei.ac.kr>

ABSTRACT

Background: EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information.

Results: Here we report for the first time the 2.1-A resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic alpha/beta hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other alpha/beta hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the beta8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1.

Conclusion: Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.

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Related in: MedlinePlus

Kinetic analysis of the thermostability of wild-type and mutant EstE1. The enzymes (6.0 μM) of wild-type EstE1 (●), EstE1F276A (■), EstE1F276E (□), EstE1V274A (▲), EstE1V274A/F276A (▯), EstE1L299D (▼), EstE1R270A (▯), EstE1E295A (▯), and EstE1R270A/E295A (▯), in 20 mM potassium phosphate buffer (pH 7.0) were incubated at 80°C for the indicated times. Residual activities were then determined by measuring the amount of p-nitrophenol released by esterase-catalyzed hydrolysis. The activity of a non-incubated sample was defined as 100%
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Figure 6: Kinetic analysis of the thermostability of wild-type and mutant EstE1. The enzymes (6.0 μM) of wild-type EstE1 (●), EstE1F276A (■), EstE1F276E (□), EstE1V274A (▲), EstE1V274A/F276A (▯), EstE1L299D (▼), EstE1R270A (▯), EstE1E295A (▯), and EstE1R270A/E295A (▯), in 20 mM potassium phosphate buffer (pH 7.0) were incubated at 80°C for the indicated times. Residual activities were then determined by measuring the amount of p-nitrophenol released by esterase-catalyzed hydrolysis. The activity of a non-incubated sample was defined as 100%

Mentions: Kinetic analyses of the thermal stability of wild-type and mutant EstE1 proteins also support the notion that the thermostability of EstE1 correlates with its ability to form a dimer (Figure 6). By mutating the sites involved in the hydrophobic interaction between two molecules of EstE1, EstE1F276E and EstE1V274A/F276A dramatically lost their enzyme activities after incubation for 1 h at 80°C. The estimated half-life values of EstE1F276E and EstE1V274A/F276A were 6.5 and 29 min, respectively. In contrast, wild-type EstE1 showed no significant decrease in activity even after incubation for 2 h. EstE1F276A, which did not exist in a monomeric form, lost approximately 50% of its activity after incubation for 2 h at 80°C, suggesting that weaker hydrophobic interactions can be disrupted during heat denaturation. This result is consistent with the thermal denaturation pattern of EstE1F276A, displaying an earlier denaturation profile compared to wild-type EstE1 (Figure 5B). EstE1L299D appeared as a dimer (Figure 4) and retained a high thermostability, indicating that Leu299 is not detrimental for EstE1 dimerization and thermostability. Introduction of mutations to the residues involved in salt bridges at the EstE1 dimer interface did not induce conversion of EstE1 dimer to a monomer form (Figure 4). EstE1R270A and EstE1R270A/E295A displayed a slight decrease in their thermostability (over 75% activity after incubation for 2 h at 80°C), and EstE1E295A still maintained its activity (Figure 6). These results suggest that salt bridges are not the major contributor required for EstE1 dimerization and thermostability, whereas two hydrophobic interfacial spots, Val274 and Phe276 on the β8 strand of two molecules of EstE1, are critical for EstE1 dimerization and thermostability.


Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties.

Byun JS, Rhee JK, Kim ND, Yoon J, Kim DU, Koh E, Oh JW, Cho HS - BMC Struct. Biol. (2007)

Kinetic analysis of the thermostability of wild-type and mutant EstE1. The enzymes (6.0 μM) of wild-type EstE1 (●), EstE1F276A (■), EstE1F276E (□), EstE1V274A (▲), EstE1V274A/F276A (▯), EstE1L299D (▼), EstE1R270A (▯), EstE1E295A (▯), and EstE1R270A/E295A (▯), in 20 mM potassium phosphate buffer (pH 7.0) were incubated at 80°C for the indicated times. Residual activities were then determined by measuring the amount of p-nitrophenol released by esterase-catalyzed hydrolysis. The activity of a non-incubated sample was defined as 100%
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1936996&req=5

Figure 6: Kinetic analysis of the thermostability of wild-type and mutant EstE1. The enzymes (6.0 μM) of wild-type EstE1 (●), EstE1F276A (■), EstE1F276E (□), EstE1V274A (▲), EstE1V274A/F276A (▯), EstE1L299D (▼), EstE1R270A (▯), EstE1E295A (▯), and EstE1R270A/E295A (▯), in 20 mM potassium phosphate buffer (pH 7.0) were incubated at 80°C for the indicated times. Residual activities were then determined by measuring the amount of p-nitrophenol released by esterase-catalyzed hydrolysis. The activity of a non-incubated sample was defined as 100%
Mentions: Kinetic analyses of the thermal stability of wild-type and mutant EstE1 proteins also support the notion that the thermostability of EstE1 correlates with its ability to form a dimer (Figure 6). By mutating the sites involved in the hydrophobic interaction between two molecules of EstE1, EstE1F276E and EstE1V274A/F276A dramatically lost their enzyme activities after incubation for 1 h at 80°C. The estimated half-life values of EstE1F276E and EstE1V274A/F276A were 6.5 and 29 min, respectively. In contrast, wild-type EstE1 showed no significant decrease in activity even after incubation for 2 h. EstE1F276A, which did not exist in a monomeric form, lost approximately 50% of its activity after incubation for 2 h at 80°C, suggesting that weaker hydrophobic interactions can be disrupted during heat denaturation. This result is consistent with the thermal denaturation pattern of EstE1F276A, displaying an earlier denaturation profile compared to wild-type EstE1 (Figure 5B). EstE1L299D appeared as a dimer (Figure 4) and retained a high thermostability, indicating that Leu299 is not detrimental for EstE1 dimerization and thermostability. Introduction of mutations to the residues involved in salt bridges at the EstE1 dimer interface did not induce conversion of EstE1 dimer to a monomer form (Figure 4). EstE1R270A and EstE1R270A/E295A displayed a slight decrease in their thermostability (over 75% activity after incubation for 2 h at 80°C), and EstE1E295A still maintained its activity (Figure 6). These results suggest that salt bridges are not the major contributor required for EstE1 dimerization and thermostability, whereas two hydrophobic interfacial spots, Val274 and Phe276 on the β8 strand of two molecules of EstE1, are critical for EstE1 dimerization and thermostability.

Bottom Line: The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other alpha/beta hydrolases.In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1.Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul, Korea. lovemilk99@yonsei.ac.kr <lovemilk99@yonsei.ac.kr>

ABSTRACT

Background: EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information.

Results: Here we report for the first time the 2.1-A resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic alpha/beta hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other alpha/beta hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the beta8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1.

Conclusion: Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.

Show MeSH
Related in: MedlinePlus