Cloning, Overexpression, and Properties of a New Thermophilic and Thermostable Esterase with Sequence Similarity to Hormone-Sensitive Lipase Subfamily from the Archaeon Archaeoglobus fulgidus Giuseppe Manco,* ,1 Elena Giosue `,† Sabato D’Auria,* , ‡ Petr Herman,‡ Giacomo Carrea,† and Mose ` Rossi* , § *Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, 80125 Naples, Italy; †Istituto di Biocatalisi e Riconoscimento Molecolare, CNR, Via Mario Bianco 9, 20131, Milano, Italy; ‡Center for Fluorescence Spectroscopy, University of Maryland at Baltimore, Baltimore, Maryland; and §Universita ` degli Studi di Napoli Federico II, Via Mezzocannone 16, 80100, Naples, Italy Received June 29, 1999, and in revised form September 9, 1999 A new esterase gene from the hyperthermophilic archaeon Archaeoglobus fulgidus, reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family, was cloned by means of the polymerase chain reaction from the A. fulgidus genome. In order to compare the biochemical properties of this putative hyperthermo- philic enzyme with those of the homologous, thermo- philic member of HSL group, namely Alicyclobacillus (formerly Bacillus) acidocaldarius esterase 2 (EST2), an overexpression system in Escherichia coli was es- tablished. The recombinant protein, expressed in sol- uble and active form at 20 mg/liter of E. coli culture, was purified to homogeneity and characterized. The enzyme, a 35.5-kDa monomeric protein, was demon- strated to be a B-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of in- hibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-hexanoate with K m and k cat values of 11  3 M (mean  SD, n  3) and 1014  38 s 1 (mean  SD, n  3), respectively, at 70°C and pH 7.1. Inactivation by diethylpyrocarbonate, phenylmethyl- sulfonylfluoride, diisopropylfosfofluoridate (DFP), and physostigmine, as well as labeling with [ 3 H]DFP, supported our previous suggestion of a catalytic triad made up of Ser 160 -His 285 -Asp 255 . The sequence identity with the thermostable A. acidocaldarius EST2 was 42.5%. The enzyme proved to be much more stable than its Alicyclobacillus counterpart. The conformational dynamics of the two proteins were investigated by frequency-domain fluorometry and anisotropy decay and the activity/stability/temperature relationship was discussed. © 2000 Academic Press Key Words: carboxylesterase; thermostability; flexi- bility; Archaeoglobus fulgidus; Alicyclobacillus acido- caldarius. Esterases, lipases, and cholinesterases belong to a large family of phylogenetically related proteins with representatives in the domains of Eukarya and Bacte- ria which include proteins lacking of enzymatic activity (1, 2). Three subfamilies have been identified: the C group includes cholinesterases from vertebrate and in- vertebrate, lipases from fungi, several esterases, and some nonenzymic proteins; the group L includes lipases from vertebrate and bacteria, lipoprotein lipases, lecithin-cholesterol acyltransferases, and re- lated nonenzymatic vitellogenins from flies; the third group was named hormone-sensitive lipase (HSL) 2 by Hemila ¨ et al. (1), since these authors reported the cloning and sequencing of a gene from Alicyclobacillus acidocaldarius encoding a protein of unknown function 1 To whom correspondence and reprint requests should be ad- dressed: Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, 80125 Naples, Italy. Fax: 81-7257240 (or 81-2396525). E-mail: manco@dafne.ibpe.na.cnr.it. 2 Abbreviations used: ORF, open reading frame; DFP, diisopropyl fluorophosphate; PMSF, phenylmethylsulphonylfluoride; DPC, di- ethylpyrocarbonate; IPTG, isopropyl -D-thiogalactoside; AFEST, A. fulgidus esterase; EST2, esterase 2; HSL, hormone-sensitive lipase; PNP, paranitrophenyl. 182 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 373, No. 1, January 1, pp. 182–192, 2000 Article ID abbi.1999.1497, available online at http://www.idealibrary.com on