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 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 hyperthermophilic enzyme with those of the homologous, thermophilic member of HSL group, namely Alicyclobacillus (formerly Bacillus) acidocaldarius esterase 2 (EST2), an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble 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 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 with K(m) and k(cat) values of 11 +/- 3 microM (mean +/- SD, n = 3) and 1014 +/- 38 s(-1) (mean +/- SD, n = 3), respectively, at 70 degrees C and pH 7.1. Inactivation by diethylpyrocarbonate, phenylmethylsulfonylfluoride, 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.
Manco
G
G
Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, Naples, 80125, Italy. manco@dafne.ibpe.na.cnr.it
Giosuè
E
E
D'Auria
S
S
Herman
P
P
Carrea
G
G
Rossi
M
M
D000595:Amino Acid Sequence; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics*; D001406:Bacillaceae / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013696:Temperature
D017931:DNA Primers; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D004950:Esterases