The crystal structure of an esterase from the hyperthermophilic microorganism Pyrobaculum calidifontis VA1 explains its enantioselectivity
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The highly thermostable esterase from the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 (PestE) shows high enantioselectivity (E > 100) in the kinetic resolution of racemic chiral carboxylic acids, but little selectivity towards acetates of tertiary alcohols (E = 2–4). To explain these unique properties, its crystal structure has been determined at 2.0 Å resolution. The enzyme is a member of the hormone-sensitive lipase group (group H) of the esterase/lipase superfamily on the basis of the amino acid sequence identity. The PestE structure shows a canonical α/β-hydrolase fold as core domain with a cap structure at the C-terminal end of the β-sheet. A tetramer in the crystal packing is formed of two dimers; the dimeric form is observed in solution. Conserved dimers and even tetramers are found in other group H proteins. The amino acid residues Ser157, His284, and Asp254 form the catalytic triad, which is typically found in α/β-hydrolases. The oxyanion hole is composed of Gly85 and Gly86 within the conserved sequence motif HGGG(M,F,W) (amino acid residues 83–87) and Ala158. With the elucidated structure, experimental results about enantioselectivity towards the two model substrate classes (as exemplified for 3-phenylbutanoic acid ethyl ester and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate) could be explained by molecular modeling. For both enantiomers of the tertiary alcohol, orientations in two binding pockets were obtained without significant energy differences corresponding to the observed low enantioselectivity due to missing steric repulsions. In contrast, for the carboxylic acid ester, two different orientations with significant energy differences for each enantiomer were found matching the high E values.
KeywordsHyperthermophilic esterase α/β-Hydrolase fold Crystal structure Enantioselectivity
We are grateful to the Deutsche Bundesstiftung Umwelt (Osnabrück, Germany) for financial support (grant no. AZ13198) and thank Britta Girbardt (Institute of Biochemistry, Greifswald) for her support in crystallization.
- Arndt JW, Schwarzenbacher R, Page R, Abdubek P, Ambing E, Biorac T, Canaves JM, Chiu HJ, Dai XP, Deacon AM, DiDonato M, Elsliger MA, Godzik A, Grittini C, Grzechnik SK, Hale J, Hampton E, Han GW, Haugen J, Hornsby M, Klock HE, Koesema E, Kreusch A, Kuhn P, Jaroszewski L, Lesley SA, Levin I, McMullan D, McPhillips TM, Miller MD, Morse A, Moy K, Nigoghossian E, Ouyang J, Peti WS, Quijano K, Reyes R, Sims E, Spraggon G, Stevens RC, van den Bedem H, Velasquez J, Vincent J, von Delft F, Wang XH, West B, White A, Wolf G, Xu QP, Zagnitko O, Hodgson KO, Wooley J, Wilson IA (2005) Crystal structure of an alpha/beta serine hydrolase (YDR428C) from Saccharomyces cerevisiae at 1.85 angstrom resolution. Proteins 58:755–758CrossRefGoogle Scholar
- Bornscheuer UT, Kazlauskas RJ (2006) Hydrolases in organic synthesis. Wiley-VCH, WeinheimGoogle Scholar
- Chen VB, Arendall WB III, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D66:12–21Google Scholar
- DeLano WL (2008) The PyMOL molecular graphics system. DeLano Scientific LLC, Palo AltoGoogle Scholar
- Duan Y, Wu C, Chowdhury S, Lee MC, Xiong G, Zhang W, Yang R, Cieplak P, Luo R, Lee T, Caldwell J, Wang J, Kollman P (2003) A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J Comput Chem 24:1999–2012CrossRefGoogle Scholar
- Hashimoto N, Aoyama T, Shiori T (1981) New methods and reagents in organic synthesis.14. A simple efficient preparation of methyl esters with trimethylsilyldiazomethane (TMSCHN2) and its application to gas chromatographic analysis of fatty acids. Chem Pharm Bull 29:1475–1478Google Scholar
- Ileperuma NR, Marshall SD, Squire CJ, Baker HM, Oakeshott JG, Russell RJ, Plummer KM, Newcomb RD, Baker EN (2007) High-resolution crystal structure of plant carboxylesterase AeCXE1, from Actinidia eriantha, and its complex with a high-affinity inhibitor paraoxon. Biochemistry 46:1851–1859CrossRefGoogle Scholar
- Leslie AGW (1992) Joint CCP4-ESF-EAMCB Newsletter on Protein Crystallography 26Google Scholar