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. 2012 Jun;80(6):1545-59.
doi: 10.1002/prot.24041. Epub 2012 Mar 13.

Functional and Structural Characterization of a Thermostable Acetyl Esterase From Thermotoga Maritima

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Free PMC article

Functional and Structural Characterization of a Thermostable Acetyl Esterase From Thermotoga Maritima

Mark Levisson et al. Proteins. .
Free PMC article

Abstract

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-β-D-xylopyranoside monoacetates as substrates in a β-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic α/β-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction.

Figures

Figure 1
Figure 1
Substrates and inhibitors of the CE7 family of enzymes. Structures of (A) acetylated xylooligosaccharide, (B) cephalosporin C, (C) p-nitrophenyl-acetate, (D) phenylmethylsulfonyl fluoride (PMSF), and (E) paraoxon.
Figure 2
Figure 2
Overall fold and topology of TM0077. (A) Stereo view of a TM0077 protomer. The β-strands are labeled numerically (-1 to 8) with the core strands in red, α-helices are labeled alphabetically (A-2 to F) and 310-helices are labeled with an Eta (η1 and η2) with the core helices in cyan. The three-helix insertion after β6 is colored green and the N-terminal extension is colored sky blue. The figure was generated using Pymol . (B) Topology diagram of TM0077, with the helices displayed as cylinders and the strands displayed as arrows following the color and label scheme of (A). The location of residues forming the catalytic triad is also indicated.
Figure 3
Figure 3
Structural superposition of TM0077 with structurally related esterases. Superposition of TM0077 (yellow) with (A) the cephalosporin C deacetylase (CAH) from B. subtilis (PDB: 1ods; blue) and (B) the α/β-hydrolase domain of the acylpeptide hydrolase/esterase apAPH from A. pernix K1 (PDB: 1ve6; grey) .
Figure 4
Figure 4
TM0077 oligomeric assembly. (A) Surface representation of the biological unit of the TM0077-Native hexamer with each monomer in a different color (left). The “cross section” shows the entrances on either side of the assembly and the internal cavity (center), and a 90° rotated view of the TM0077-Native hexamer, with a close-up view of the open central hole (right). (B) Surface representation of the biological hexamer unit of CAH from B. subtilis (left) and the TM0077-SeMet hexamer with a close-up view of the blocked central hole (right).
Figure 5
Figure 5
Effect of temperature and pH on esterase activity. (A) The esterase activity was studied using pNP-C2 as a substrate at temperatures ranging from 40–100°C. The inset shows the temperature dependence as an Arrhenius plot. (B) Thermal stability of TM0077 at 90°C. (C) The effect of pH on esterase activity studied using pNP-C2 as a substrate at pH values in the range of 4.8–9.2.
Figure 6
Figure 6
TM0077 catalytic site. (A) Surface representation of the TM0077 catalytic site, with His303, Asp274 and the intermediate DEP-modified Ser188 shown as sticks. The two binding pockets are indicated with S1 and S2. (B) Apo TM0077 with a bound chloride ion (green sphere), (C) TM0077 with PMS-modified Ser188 and (D) TM0077 with DEP-modified Ser188. The catalytic residues are shown as sticks, with the hydrogen bonds shown as dashed lines. Carbon atoms are in green (apo), cyan (PMS) or blue (DEP), oxygen atoms in red, sulfur atoms in yellow and phosphate in orange. Electron density omit maps shown for inhibitor modified Ser188 contoured at 1σ show that the PMS and DEP are covalently bonded to Ser188 in (C) and (D), respectively. Distances are shown in Ångströms.
Figure 7
Figure 7
Conformational change of Ser188 Oγ. The Oγ atom of the Ser188 is rotated ~110° between the native apo structure (cyan) and (A) the complexed PMS-modified Ser188 structure (pink), (B) the DEP-modified Ser188 structure (light blue) and (C) the SeMet structure (purple). The different hydrogen bonds made for the Ser Oγ in the native versus complexed structures are shown as dashed black lines with distances in Ångströms.

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