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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

Analysis of wild-type and mutant EstE1 by gel filtration chromatography. Purified EstE1 proteins were loaded onto a Superdex 200 column and eluted as described in Methods. Estimated molecular weights of wild-type EstE1 (●), EstE1F276A (■), EstE1F276E (□), EstE1V274A (▲), EstE1V274A/F276A (▯), EstE1L299D (▼), EstE1R270A (▯), EstE1E295A (▯), and EstE1R270A/E295A (▯) are presented. Molecular mass standards (albumin, 67 kDa; ovalbumin, 43 kDa; and chymotrypsinogen A, 25 kDa) were subjected to the same process, and their migration is indicated.
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Figure 4: Analysis of wild-type and mutant EstE1 by gel filtration chromatography. Purified EstE1 proteins were loaded onto a Superdex 200 column and eluted as described in Methods. Estimated molecular weights of wild-type EstE1 (●), EstE1F276A (■), EstE1F276E (□), EstE1V274A (▲), EstE1V274A/F276A (▯), EstE1L299D (▼), EstE1R270A (▯), EstE1E295A (▯), and EstE1R270A/E295A (▯) are presented. Molecular mass standards (albumin, 67 kDa; ovalbumin, 43 kDa; and chymotrypsinogen A, 25 kDa) were subjected to the same process, and their migration is indicated.

Mentions: The residues responsible for dimerization of EstE1 subunits differ from those of AFEST. A previous study showed that intermolecular contacts present in AFEST consisted of hydrogen bonds and salt bridges [19]. Amino acid sequence alignment revealed that Val274, Phe276, and Leu299 of EstE1 correspond to Val278, Tyr280, and Gln303 of AFEST, respectively (Figure 3), but these residues in AFEST did not participate in similar hydrophobic interactions as observed in EstE1. Instead, hydrogen bonds between Tyr280A-Gln303B and Tyr280B-Gln303A, and weak hydrophobic interactions between Val278A and Val278B, appear to contribute to the intermolecular interactions observed in AFEST (Figure 2E). To verify the roles of the hydrophobic interactions and salt bridges identified in EstE1 dimer interface, eight EstE1 mutant proteins containing single or multiple amino acid substitutions were created. Five EstE1 mutants had disruptions in hydrophobic interactions. Val274 was replaced with Ala to generate EstE1V274A. Phe276 was replaced with Ala and Glu to generate EstE1F276A and EstE1F276E, respectively. Leu299 was changed to Asp in EstE1L299D. Both Phe276 and Val274 were changed to Ala in EstE1V274A/F276A. In addition, three EstE1 mutants containing changes in the residues involved in the salt bridges were generated. Arg270 and Glu295 residues were replaced with Ala to generate EstE1R270A and EstE1E295A, respectively. Both of these amino acids were changed to Ala to generate EstE1R270A/E295A. All of these mutants were expressed in E. coli and purified to near-homogeneity from cell extracts that were not heat-treated, using Ni-affinity chromatography as described previously [8]. Analysis of the molecular weight of the purified mutant proteins by gel filtration chromatography (GFC) revealed that EstE1F276E and EstE1V274A/F276A converted to a monomer of approximately 42 and 38 kDa, respectively, while wild-type EstE1 and other mutants were the size of a dimer with a molecular weight of ~60 kDa (Figure 4). EstE1F276A remained as a dimer whereas a single mutation of Phe to Glu in EstE1F276E completely abolished dimerization. This is likely due to the fact that Ala has a similar hydrophobicity scale as that of Phe such that EstE1 molecules maintained their hydrophobic interactions, while change of the Phe to hydrophilic residue Glu did not permit the formation of hydrophobic interactions with Val274 in another molecule of EstE1. This result underscores the critical role of hydrophobic interactions through Phe276. In addition, even though a single amino acid change in EstE1V274A and EstE1F276A did not convert them to monomers, the combination of these two mutations in EstE1V274A/F276A abolished its ability to form dimers, suggesting the hydrophobic interactions involving both Val274 and Phe276 are important for EstE1 dimerization. Likewise, native PAGE analysis revealed a faster electrophoretic mobility of EstE1F276E and EstE1V274A/F276A compared to that of wild-type EstE1 (data not shown). In contrast, the EstE1 mutant proteins with Ala substitution of Arg270 or Asp295 (EstE1R270A and EstE1E295A), and the mutant with Ala substitution on both of these residues (EstE1R270A/E295A) were still able to dimerize, indicating salt bridges at the both ends of the hydrophobic interface are not critical for EstE1 dimerization.


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)

Analysis of wild-type and mutant EstE1 by gel filtration chromatography. Purified EstE1 proteins were loaded onto a Superdex 200 column and eluted as described in Methods. Estimated molecular weights of wild-type EstE1 (●), EstE1F276A (■), EstE1F276E (□), EstE1V274A (▲), EstE1V274A/F276A (▯), EstE1L299D (▼), EstE1R270A (▯), EstE1E295A (▯), and EstE1R270A/E295A (▯) are presented. Molecular mass standards (albumin, 67 kDa; ovalbumin, 43 kDa; and chymotrypsinogen A, 25 kDa) were subjected to the same process, and their migration is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Analysis of wild-type and mutant EstE1 by gel filtration chromatography. Purified EstE1 proteins were loaded onto a Superdex 200 column and eluted as described in Methods. Estimated molecular weights of wild-type EstE1 (●), EstE1F276A (■), EstE1F276E (□), EstE1V274A (▲), EstE1V274A/F276A (▯), EstE1L299D (▼), EstE1R270A (▯), EstE1E295A (▯), and EstE1R270A/E295A (▯) are presented. Molecular mass standards (albumin, 67 kDa; ovalbumin, 43 kDa; and chymotrypsinogen A, 25 kDa) were subjected to the same process, and their migration is indicated.
Mentions: The residues responsible for dimerization of EstE1 subunits differ from those of AFEST. A previous study showed that intermolecular contacts present in AFEST consisted of hydrogen bonds and salt bridges [19]. Amino acid sequence alignment revealed that Val274, Phe276, and Leu299 of EstE1 correspond to Val278, Tyr280, and Gln303 of AFEST, respectively (Figure 3), but these residues in AFEST did not participate in similar hydrophobic interactions as observed in EstE1. Instead, hydrogen bonds between Tyr280A-Gln303B and Tyr280B-Gln303A, and weak hydrophobic interactions between Val278A and Val278B, appear to contribute to the intermolecular interactions observed in AFEST (Figure 2E). To verify the roles of the hydrophobic interactions and salt bridges identified in EstE1 dimer interface, eight EstE1 mutant proteins containing single or multiple amino acid substitutions were created. Five EstE1 mutants had disruptions in hydrophobic interactions. Val274 was replaced with Ala to generate EstE1V274A. Phe276 was replaced with Ala and Glu to generate EstE1F276A and EstE1F276E, respectively. Leu299 was changed to Asp in EstE1L299D. Both Phe276 and Val274 were changed to Ala in EstE1V274A/F276A. In addition, three EstE1 mutants containing changes in the residues involved in the salt bridges were generated. Arg270 and Glu295 residues were replaced with Ala to generate EstE1R270A and EstE1E295A, respectively. Both of these amino acids were changed to Ala to generate EstE1R270A/E295A. All of these mutants were expressed in E. coli and purified to near-homogeneity from cell extracts that were not heat-treated, using Ni-affinity chromatography as described previously [8]. Analysis of the molecular weight of the purified mutant proteins by gel filtration chromatography (GFC) revealed that EstE1F276E and EstE1V274A/F276A converted to a monomer of approximately 42 and 38 kDa, respectively, while wild-type EstE1 and other mutants were the size of a dimer with a molecular weight of ~60 kDa (Figure 4). EstE1F276A remained as a dimer whereas a single mutation of Phe to Glu in EstE1F276E completely abolished dimerization. This is likely due to the fact that Ala has a similar hydrophobicity scale as that of Phe such that EstE1 molecules maintained their hydrophobic interactions, while change of the Phe to hydrophilic residue Glu did not permit the formation of hydrophobic interactions with Val274 in another molecule of EstE1. This result underscores the critical role of hydrophobic interactions through Phe276. In addition, even though a single amino acid change in EstE1V274A and EstE1F276A did not convert them to monomers, the combination of these two mutations in EstE1V274A/F276A abolished its ability to form dimers, suggesting the hydrophobic interactions involving both Val274 and Phe276 are important for EstE1 dimerization. Likewise, native PAGE analysis revealed a faster electrophoretic mobility of EstE1F276E and EstE1V274A/F276A compared to that of wild-type EstE1 (data not shown). In contrast, the EstE1 mutant proteins with Ala substitution of Arg270 or Asp295 (EstE1R270A and EstE1E295A), and the mutant with Ala substitution on both of these residues (EstE1R270A/E295A) were still able to dimerize, indicating salt bridges at the both ends of the hydrophobic interface are not critical for EstE1 dimerization.

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