Istituto di Biochimica delle Proteine ed Enzimologia, Via Marconi 10, 80125 Naples, Italy
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Abstract |
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Keywords: affinity labeling/Archaeoglobus fulgidus/catalytic triad/homology modeling/thermostable carboxylesterase
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Introduction |
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Recently we reported the purification of a new esterase (EST2) from A.acidocaldarius and demonstrated its identity with the aforementioned ORF3. On the basis of a secondary structure-driven multiple sequence alignment we predicted for this enzyme the /ß-hydrolase topological fold common to several esterases and lipases and identified Ser155, Asp252 and His282 as the putative members of the catalytic triad (Manco et al., 1997
). The gene was over-expressed in Escherichia coli and the protein was purified and characterized to obtain further insights into the structurefunction relationship of this interesting representative of the HSL group (Manco et al., 1998a
). The enzyme has been crystallized (De Simone et al., 1999
) and resolution of the 3D structure is currently under way. This will permit the comparison with the recently solved structure of brefeldin A esterase (BFAE) from Bacillus subtilis, a mesophilic representative of the HSL group (Wei et al., 1999
).
More recently, the ORF AF1763 identified in Archeoglobus fulgidus as a hypothetical esterase (AFEST) (Klenk et al., 1997) has been overexpressed in E.coli, purified and demonstrated by us to be a hyper-thermophilic carboxylesterase (Manco et al., 2000
). In this work we exploited a combined approach of affinity labeling and molecular modeling to investigate the structural features of the enzyme.
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Materials and methods |
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AFEST and BFAE sequence alignment was obtained from a secondary structure-driven multisequence alignment of the HSL group (not shown) using the Clustal W program (Thompson et al., 1994) and refined manually to reduce to the minimum gaps which were not allowed inside the structural elements.
Molecular modeling was performed on a Silicon Graphics O2 workstation using the commercial software packages InsightII (InsightII User Guide, October 1995, Biosym/MSI, San Diego, CA).
The high-resolution X-ray crystal structure of BFAE was used as a template structure. Several 3D models were constructed using the Modeler module (Sali et al., 1995) within InsightII. The methodology is based on satisfaction of spatial restraints that are obtained from an alignment of a target sequence with related 3D structures at high resolution (1.82.4 Å), using a conjugate gradient and a molecular dynamics-simulated annealing as optimization procedures. Each model was also opportunely minimized using the Discover3 module within InsightII.
The resulting models were verified using the on-line software WHAT IF at http://biotech.ebi.ac.uk:8400/chk/whatif/index.html.
Enzyme labeling with [3H]diisopropylphosphofluoridate [3H]DFP
Protein expression and purification have been described elsewhere (Manco et al., 2000).
A sample of 350 µg of pure protein (11 nmol) was labeled by incubation with 140 µCi of [3H]DFP (23 nmol; 60x106 c.p.m.) in 300 µl of 20 mM TrisHCl buffer (pH 8.0) at 37°C for 2 h and then overnight at room temperature. The unreacted inhibitor was separated from protein by gel filtration through Sephadex G-25 equilibrated and eluted with 20 mM TrisHCl (pH 8.0) containing 1 mM MgCl2, 0.5 mM EDTA, 100 mM NaCl. Fractions of 0.5 ml were collected and radioactivity eluting at the void volume was measured in a Packard Tri-Carb 300 liquid scintillation counter. Radioactive fractions were pooled and enzyme activity and radioactivity were measured. The radioactivity and enzyme activity recoveries were 50% and 13%, respectively.
Protein alkylation and digestion
The radioactively labeled protein (175 µg, corresponding to 5.5 nmol of protein) was lyophilized, dissolved in denaturing buffer containing 0.5 M TrisHCl (pH 8.0), 2 mM EDTA, 6 M guanidine-HCl and reduced with dithiothreitol (100 nmol) for 3 h at 37°C under nitrogen. 4-Vinylpyridine (9 µmol) was added and the reaction proceeded for 45 min at room temperature in the dark under nitrogen. The protein was immediately desalted by gel filtration on a PD-10 column (Pharmacia, Uppsala, Sweden) equilibrated in 0.1% trifluoroacetic acid and lyophilized. The lyophilized protein was resuspended in 25 mM TrisHCl (pH 8.5) containing 10% acetonitrile and 1 mM EDTA and digested with 5 µg of endoproteinase Lys-C (Boheringer, Mannheim, Germany) for 4 h at 37°C. The peptide mixture was separated by reversed-phase HPLC on a C18 Vydac column with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. The absorbance at 220 nm and 3H radioactivity were monitored.
Amino acid sequencing
Automated Edman degradation was performed on a Procise Protein Sequencer Model 492, equipped with a 140C Microgradient System from Perkin-Elmer (Applied Biosystems Division). Fractions were collected at each cycle, after HPLC elution of the released phenylthiohydantoin amino acid and aliquots were counted for 3H radioactivity.
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Results and discussion |
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To test further the role of Ser160 as the nucleophilic element of the triad we used a biochemical approach, as follows. The pure enzyme was labeled as described previously (Manco et al., 1998a) with [3H]DFP which totally inhibits the activity of serine-type enzymes by irreversible phosphorylation of the essential nucleophilic residue; it was made free of unreacted inhibitor by gel filtration on a Sephadex G-25 column and subjected to protease digestion in order to identify the labeled peptide and the reactive serine residue. After endoproteinase Lys-C digestion and HPLC separation of the resulting peptide mixture, most of the radioactivity was found associated with a single peak (Figure 3
). Amino acid sequencing revealed that the fraction contained two co-eluting peptides. Taking advantage of the different amounts of the peptides in the mixture, simultaneous sequencing was carried out. Twenty-five cycles of Edman degradation yielded the sequences (i) MLDMPIDPVYYQLAEYFDSLPK and (ii) IFVGGD(S)AGGNLAAAVSIMARDSGE and the peptides were identified on the basis of the sequence deduced from DNA. The first peptide corresponded to the amino-terminal region including the N-terminal methionine (positions 122). The second peptide corresponded to positions 154178 of the total sequence. The [3H]DFP-modified serine residue was identified by the appearance of radioactivity in the fractions released following sequential degradation and was localized at the seventh cycle where Ser160 of the 154178 peptide was expected to be eluted (Figure 4
). At this cycle, a very low signal for the serine residue could be detected (data not shown). Since the [3H]DFP-modified serine is expected to be eluted at a different position from that of serine, the small amount of serine detected at this cycle was probably due to partial cleavage of the diisopropyl moiety from the labeled serine residue during the routine of Edman degradation. Serine residues not corresponding to radioactive signals were also detected at the seventeenth and twenty-third cycles corresponding to positions 170 and 176 of the 154178 peptide and at nineteenth cycle corresponding to position 19 of the 122 peptide. This result together with the information resulting from the 3D model demonstrate conclusively that Ser160 is the nucleophilic member of the catalytic triad in AFEST.
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This study set up the guidelines for a deeper analysis of the relationship between the structure and function of a A.fulgidus esterase. The 3D model will provide a 3D framework for the rational design of site-specific mutations to address the problem of protein thermophilicity, thermostability and substrate specificity before we succeed in the resolution of the 3D structure for this and the related thermophilic eubacterial EST2 from A.acidocaldarius. The fact that these two enzymes share many features and are practically colinear in sequence (Manco et al, 2000) and yet are derived from organisms in different domains of life is noteworthy, since it speaks of the probable gene transfer that may account for the similarity. Even more intriguing is the similarity of these enzymes with two members of the HSL group (Moraxella and human lipases) showing adaptation to low temperatures (Feller et al., 1991
; Langin et al, 1993
). We hope that the accumulation of new data on these and other enzymes in the HSL group will accelerate the understanding of the evolutive and structurefunction relationiships in this interesting group of enzymes.
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Acknowledgments |
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Notes |
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References |
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Received August 2, 1999; revised November 29, 1999; accepted December 8, 1999.