Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus

A new esterase gene from the hyperthermophilic archaeon Archaeoglobus fulgi dus, 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 hyperthermophilic enzyme with t hose of the homologous, thermophilic member of HSL group, namely Alicycloba cillus (formerly Bacillus) acidocaldarius esterase 2 (EST2), an overexpress ion system in Escherichia coli was established. The recombinant protein, ex pressed in soluble and active form at 20 mg/liter of E, coli culture, was p urified to homogeneity and characterized. The enzyme, a 35.5-MDa monomeric protein, was demonstrated to be a B "-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP.hexanoate wit h K-m and k(cat) values of 11 +/- 3 mu M (mean +/- SD, n = 3) and 1014 +/- 38 s(-1) (mean +/- SD, n = 3), respectively, at 70 degrees C and pH 7.1. In activation by diethylpyrocarbonate, phenylmethyl-sulfonylfluoride, diisopro pylfosfofluoridate (DFP), and physostigmine, as well as labeling with [H-3] 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. acid ocaldarius EST2 was 42.5%. The enzyme proved to be much more stable than it s Alicyclobacillus counterpart. The conformational dynamics of the two prot eins were investigated by frequency-domain fluorometry and anisotropy decay and the activity/stability/temperature relationship was discussed. (C) 200 0 Academic Press.