ABBS 2005,37(09): Expression, Purification and Crystal Structure of a Truncated Acylpeptide Hydrolase from Aeropyrum pernix K1

Research Paper

Acta Biochim Biophys Sin 2005,37:613-617

doi:10.1111/j.1745-7270.2005.00085.x

Expression, Purification and Crystal Structure of a Truncated Acylpeptide Hydrolase from Aeropyrum pernix K1

Hai-Feng ZHANG, Bai-Song ZHENG1, Ying PENG, Zhi-Yong LOU, Yan FENG1*, and Zi-He RAO*

Laboratory of Structural Biology, Department of Biological Sciences and Biotechnology and Protein Sciences Laboratory of Ministry of Education, Tsinghua University, Beijing 100084, China

1 Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, China

Received: March 20, 2005

Accepted: June 7, 2005

*Corresponding authors:

Yan FENG: Tel, 86-431-8987975; E-mail, [email protected]

Zi-He RAO: Tel, 86-10-62771493; Fax, 86-10-62773145; E-mail, [email protected]

Abstract Acylpeptide hydrolase (APH) catalyzes the N-terminal hydrolysis of Na-acylpeptides to release Na-acylated amino acids. The crystal structure of recombinant APH from the thermophilic archaeon Aeropyrum pernix K1 (apAPH) was reported recently to be at a resolution of 2.1 Å using X-ray diffraction. A truncated mutant of apAPH that lacks the first short a-helix at the N-terminal, apAPH-D(1-21), was cloned, expressed, characterized and crystallized. Data from biochemical experiments indicate that the optimum temperature of apAPH is decreased by 15 ºC with the deletion of the N-terminal a-helix. However, the enzyme activity at the optimal temperature does not change. It suggests that this N-terminal a-helix is essential for thermostability. Here, the crystal structure of apAPH-D(1-21) has been determined by molecular replacement to 2.5 Å. A comparison between the two structures suggests a difference in thermostability, and it can be concluded that by adding or deleting a linking structure (located over different domains), the stability or even the activity of an enzyme can be modified.

Key words acylpeptide hydrolase; Aeropyrum pernix K1; crystal structure

Acylpeptide hydrolase (APH; also known as acylamino acid-releasing enzyme or acylaminoacyl peptidase [EC3.4.19.1]), a member of the prolyl oligopeptidase (POP) family of serine proteases, catalyzes the N-terminal hydrolysis of Na-acylpeptides to release Na-acylated amino acids [1]. In addition to APH, the members of the prolyl oligopeptidase family include dipeptidyl peptidase IV (DPP IV), fibroblast activation protein a, DPP7, DPP8, DPP9, prolyl carboxypeptidase, oligopeptidase B and prolyl oligopeptidase. Most of them are important in the research for new drugs [2,3].

To date, rat, porcine, human and bovine APH from various tissues have been characterized. They all consist of 732 amino acid residues, and were reported to form homotetramers [48]. However, Arabidopsis thaliana APH is a 764-amino acid protein, which exhibits 31.8% sequence identity with rat APH, and it also forms a tetramer [9]. An APH from the thermophilic archaeon Pyrococcus horikoshii OT3 has also been characterized. Being different from its mammalian counterparts, it is 100 residues shorter and forms a homodimer [10]. Inhibition of APH activity leads to apoptosis [11], and a deficiency in human APH is reported to be linked to small-cell lung carcinoma and renal cell carcinoma [1214]. APH in the porcine brain may be the target of the cognitive-enhancing effects of certain organophosphorus compounds [15].

Like P. horikoshii OT3, Aeropyrum pernix K1 grows in the temperature range of 90 to 98 ºC, with an optimal temperature of 95 ºC. However, P. horikoshii OT3 is an anaerobic euryarchaeota, and A. pernix K1 is an aerobic strain classified as crenarchaeota. The complete genome of A. pernix K1 has been sequenced, and four genes (Ape1547, Ape1832, Ape2290 and Ape2441) have been designated as encoding APHs [16]. Recently, the crystal structure of an APH from A. pernix K1 (apAPH), the gene product of Ape1547, was determined [17]. This is the first confirmed structure of an APH. Formerly, only human APH has been crystallized, but the structure remains unknown [18].

apAPH is a symmetric homodimer, and each monomer comprises two domains. The N-terminal domain is a seven-bladed b-propeller and the C-terminal catalytic domain has a canonical a/b hydrolase fold [17]. A short a-helix at the N-terminus (residues 823) extends from the b-propeller domain and forms part of the hydrolase domain; this forms a linking structure between these two domains.

To investigate how this N-terminal a-helix affects the whole structure, enzyme activity and thermostability, a mutant of apAPH in which the first 21 amino acids at the N-terminus were removed, apAPH-D(1-21), was expressed in Escherichia coli, and its crystal structure was determined.

Materials and Methods

Materials

The expression vector pET11a-apAPH-D(1-21) was constructed by the Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, Jilin University (Changchun, China). The E. coli BL21(DE3) strain was obtained from Novagen (Darmstadt, Germany, and chromatography columns were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). General reagents were of analytical grade.

Protein expression and purification

The expression vector pET11a-apAPH-D(1-21) was transformed into the E. coli BL21(DE3) strain. The conditions for expression were as follows. Bacteria were grown in LB medium (10 mg/ml tryptone, 5 mg/ml yeast extract, 10 mg/ml NaCl) containing 100 mg/ml penicillin at 37 ºC until A600=0.6, then induced with 0.5 mM isopropyl-b-D-thiogalactopyranoside (IPTG). After treatment at 25 ºC overnight, the cells were harvested by centrifugation at 5000 rpm for 10 min, resuspended in 25 mM Tris-HCl (pH 8.0) and 50 mM NaCl, and lysed by sonication. The soluble cell lysate obtained by centrifugation at 15,000 rpm for 30 min was heated at 75 ºC for 1 h. Then the supernatant obtained by centrifugation was loaded onto a Q Sepharose fast flow column previously equilibrated with 25 mM Tris-HCl (pH 8.0) containing 50 mM NaCl. After washing away the unbound protein with two bed volumes of 25 mM Tris-HCl (pH 8.0), a linear gradient of 0.051 M NaCl in the same buffer was applied. The collected fractions were centrifuged for 30 min at 15,000 rpm. The supernatant was then loaded onto a Resource Q anion-exchange chromatography column equilibrated with 25 mM Tris-HCl (pH 8.0) containing 50 mM NaCl. The eluted sample was concentrated using Filtron 5K to 250 ml and loaded onto a Superdex 75 size-exclusion chromatography column. The fraction showing only one protein band with a molecular weight of about 60 kDa after SDS-PAGE was collected and its purity was determined to be suitable for crystallization.

Crystallization

The purified protein was concentrated using Filtron 5K to 25 mg/ml in a solution containing 50 mM NaCl. apAPH crystallization conditions were used for the initial screening using the hanging-drop vapor diffusion method. The mixed drop including 1.5 ml of protein solution and 1.5 ml of reservoir solution was grown in an airtight system containing 0.4 ml of reservoir solution at 18 ºC. Crystals appeared after one month with the protein concentration higher than 25 mg/ml. The best crystallization results were obtained with 4.5%-5% (W/V) PEG 4000, 0.025 M sodium acetate, pH 4.6. To improve the quality of the crystals, 0.025%0.05% agarose gel was added.

Data collection and processing

The data were collected to 2.5 Å using a 345 mm MAR research image-plate system mounted on a Rigaku RU-2000 Cu Ka rotating-anode generator (Norderstedt, Germany) operated at 48 kV and 98 mA. During data collection, the crystal was maintained at 100 K using a Cryostream (Oxford Cryosystems, Oxford, UK) in a cryoprotectant prepared by adding 20% glycerol to the mother liquor. The data were processed and scaled with DENZO and SCALEPACK.

Results

Structure determination and refinement

The asymmetric unit of the crystal contains two molecules (denoted A and B) with an approximate solvent content of 44%. The structure of apAPH-D(1-21) was determined by molecular replacement with the program AMoRe, using the known structure of apAPH (PDB code 1VE6) as a search model. The program O was used for viewing electron density maps and manual building. Crystallography & NMR system (CNS) was used for refinement, with iterative cycles of simulated annealing and individual B-factor refinement in the resolution range of 502.5 Å. The final structure had Rwork=22.9% (Rfree=24.8%), and consisted of 561 residues in molecule A and molecule B respectively and 579 water molecules. From the Ramachandran plot generated by PROCHECK, the final structure was found to have good stereochemistry, with 73.8% of residues in the most favored region, 26% of residues in the additionally allowed region and 0.2% of residues in the generously allowed region. The final refinement statistics for the model are given in Table 1.

Comparison with apAPH

Enzymatic assay data indicate that the optimum temperature of apAPH is decreased by 15 ºC (from 90 ºC to 75 ºC) with the deletion of the N-terminal a-helix, but the enzyme activity at the optimal temperature does not change (data not shown). It suggests that this a-helix is essential for thermostability. Fig. 1 shows the superposition of the Ca backbones of apAPH-D(1-21) and wild-type apAPH. The main-chain conformations of them are very similar, except for the a-helix at the N-terminus. The root mean square (r.m.s.) deviation of all the main-chain atoms (calculated against molecule A) between the two structures is 0.873 Å. When the two domains are calculated separately, the r.m.s. deviations are 0.801 Å for the N-terminal domain and 0.829 Å for the C-terminal domain. It can be inferred that there are further differences between the C-terminal catalytic domains of two structures.

The structure of apAPH-D(1-21) is more flexible. The average B-factor values are 32.5 Å2 for apAPH-D(1-21) and 19.4 Å2 for apAPH respectively. It should be noted that the temperature factors are considerably higher for apAPH-D(1-21), indicating greater mobility along the polypeptide chain in this structure (Fig. 2).

Analysis of the electrostatic surface of apAPH-D(1-21) revealed that an additional cavity forms where the N-terminal a-helix existed (Fig. 3). It is believed that the cavity in the electrostatic surface is a disadvantage for protein stability.

Discussion

apAPH is a bifunctional enzyme, acting as both APH and esterase. Esterases have developed into the most widely used classes of enzymes in various industrial processes, including stereospecific hydrolysis, transesterification, ester synthesis and modification of physicochemical properties of triglycerides for fat biosynthesis and other organic biosynthesis reactions. However, mesophilic enzymes are often not well suited for the harsh reaction conditions, such as high temperature, exposure to organic solvents, etc., in industrial processes because of the lack of enzyme stability. The discovery of a specific esterase able to function under extreme conditions is important and will widely extend the range of reactions in which esterase can be used. Experiments indicate that apAPH has esterase activity in the temperature range of 55 to 95 ºC, with maximum activity and highest thermostability at 90 ºC (data not shown).

In general, several factors are known to affect the stability of thermophilic proteins. Primary sequence composition, salt bridge/ion pairs and hydrogen bonds, hydrophobic interaction contact area and solvent accessible surface area all contribute to thermostability. In this study, a specific method has been applied and as a result of this, a new method of changing the thermostability of an enzyme to a great extent has been discovered.

In contrast with the percentage of the first 21 amino acids in the total of 582 amino acids (3.6%), the percentages of charged and hydrophobic residues in the truncated sequence are 5.1% and 5.2% respectively. Macroscopically, an increase in the number of charged and hydrophobic residues can enhance thermostability. In addition, there are two important salt bridges (D15-R355 and R18-D325) which also improve the thermal stability. In general, it is believed that a decrease in the solvent-accessible surface area is favorable for protein stability, and the larger cavity results in an increase in solvent accessibility of the structure and thus reduces thermostability. But these values are 20,955 Å2 for apAPH and 20,289 Å2 for apAPH-D(1-21). Therefore, the cavity may influence thermostability in other ways.

In conclusion, the decrease in thermostability appears to the result of amino acid composition, fewer ionic interactions and a larger cavity in the structure. Together, these structural features may serve to enhance the thermostability of apAPH. However, as the N-terminal a-helix has no effect on the active site, the enzyme activity of apAPH-D(1-21) is the same as that of the wild-type apAPH. Moreover, the structural comparison above suggests that we can change the stability of an enzyme and at the same time retain its activity by adding or deleting a linking structure (located over different domains). A quick modification based on structure is thus possible.

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