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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

Three-dimensional structure of EstE1. A ribbon diagram of EstE1 shows the eight α-helices and eight β-strands that form a classical α/β hydrolase fold [17]. The α-helical segments and β-strands are shown in blue and yellow, respectively. G2 and G3 represent 310-helices. Helix α1 is not shown because of its disordered electron map. The catalytic triad containing residues Ser154, Asp251, and His281, are shown in stick representation. N and C denote the N and C termini, respectively.
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Figure 1: Three-dimensional structure of EstE1. A ribbon diagram of EstE1 shows the eight α-helices and eight β-strands that form a classical α/β hydrolase fold [17]. The α-helical segments and β-strands are shown in blue and yellow, respectively. G2 and G3 represent 310-helices. Helix α1 is not shown because of its disordered electron map. The catalytic triad containing residues Ser154, Asp251, and His281, are shown in stick representation. N and C denote the N and C termini, respectively.

Mentions: The final structure of EstE1 displayed a nearly ellipsoidal shape with approximate dimensions of 46 Å × 47 Å × 57 Å (Figure 1). It has the classical features of an α/β hydrolase fold [17] consisting of a central eight-stranded mixed β-sheet surrounded by the five helices α3 (Asp93-Ser103), α4 (Thr122-Leu141), α5 (Ala155-Ser170), α8 (Leu253-Ser267), and α9 (Asp291-Leu308). The electron density of the N-terminal 16 residues, which is expected to include the α1 (Lys8-Arg13) helix, was disordered. Strands β1 and β3 – β8 are parallel but the β2 strand is antiparallel to the others. The β1 and β8 strands are rotated approximately 90° because β2–β7 sheets are in the counter-clockwise direction starting from the position of the β1 strand and extending to that of the β8 strand. These β strands form an α/β fold core domain, which is the canonical architecture of an α/β hydrolase [17]. EstE1 also contains a similar cap domain present in other members of the HSL family [18-20]. The cap domain consists of two separated helical regions (containing residues 1–41 and 193–218) and locates to the upper region of the central β-sheet. The helices α1 (not shown in Figure 1; residues 9–14), α2 (residues 23–41), α6 (residues 193–200), α7 (residues 208–218), two 310-helices (G2 and G3), and several random coils between these helices constitute the cap domain.


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)

Three-dimensional structure of EstE1. A ribbon diagram of EstE1 shows the eight α-helices and eight β-strands that form a classical α/β hydrolase fold [17]. The α-helical segments and β-strands are shown in blue and yellow, respectively. G2 and G3 represent 310-helices. Helix α1 is not shown because of its disordered electron map. The catalytic triad containing residues Ser154, Asp251, and His281, are shown in stick representation. N and C denote the N and C termini, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Three-dimensional structure of EstE1. A ribbon diagram of EstE1 shows the eight α-helices and eight β-strands that form a classical α/β hydrolase fold [17]. The α-helical segments and β-strands are shown in blue and yellow, respectively. G2 and G3 represent 310-helices. Helix α1 is not shown because of its disordered electron map. The catalytic triad containing residues Ser154, Asp251, and His281, are shown in stick representation. N and C denote the N and C termini, respectively.
Mentions: The final structure of EstE1 displayed a nearly ellipsoidal shape with approximate dimensions of 46 Å × 47 Å × 57 Å (Figure 1). It has the classical features of an α/β hydrolase fold [17] consisting of a central eight-stranded mixed β-sheet surrounded by the five helices α3 (Asp93-Ser103), α4 (Thr122-Leu141), α5 (Ala155-Ser170), α8 (Leu253-Ser267), and α9 (Asp291-Leu308). The electron density of the N-terminal 16 residues, which is expected to include the α1 (Lys8-Arg13) helix, was disordered. Strands β1 and β3 – β8 are parallel but the β2 strand is antiparallel to the others. The β1 and β8 strands are rotated approximately 90° because β2–β7 sheets are in the counter-clockwise direction starting from the position of the β1 strand and extending to that of the β8 strand. These β strands form an α/β fold core domain, which is the canonical architecture of an α/β hydrolase [17]. EstE1 also contains a similar cap domain present in other members of the HSL family [18-20]. The cap domain consists of two separated helical regions (containing residues 1–41 and 193–218) and locates to the upper region of the central β-sheet. The helices α1 (not shown in Figure 1; residues 9–14), α2 (residues 23–41), α6 (residues 193–200), α7 (residues 208–218), two 310-helices (G2 and G3), and several random coils between these helices constitute the cap domain.

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