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ISSN 0582-9879                                 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(5): 569-572                                     CN 31-1300/Q

 

Short Communication

Expression, Purification, Crystallization and Preliminary X-ray Diffraction Analysis of the Mutant Pro229Ser of Thermostable Catechol 2,3-dioxygenase

JIANG Tao, JI Chao-Neng, SHENG Xiao-Yu, MAO Yu-Min*

( State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, China )

 

Abstract  The mutant Pro229Ser of thermostable catechol 2,3-dioxygenase (TC23O) was expressed and purified. Enzymatic analysis revealed that its thermostability was decreased, the temperature corresponding to 50% enzyme activity being about 10.2 ℃ lower than that of the wild type TC23O. Its kinetic parameter kcat/Km value (4.89 ×106 mol–1·s–1) was lower than that of the wild type TC23O(6.97×106 mol–1·s–1). By the hanging-drop vapor-diffusion method using polyethylene glycol 400 as a precipitant, the mutant Pro229Ser of TC23O crystallized at 4 ℃. X-ray diffraction analysis revealed that the crystals belong to the orthorhombic space group I222 with unit-cell parameters a＝7.059 nm, b＝7.415 nm, c＝13.311 nm, and they diffracted to at least 0.24 nm resolution. Assuming the presence of 2 molecules of the mutant Pro229Ser in the asymmetric unit, the Matthews parameter (Vm) was calculated to be 2.49×10-3 nm3·D–1, and the solvent content was about 51%. The crystal structure determination is now in progress.

Key words    thermostable enzyme; X-ray diffraction analysis; thermostability; thermostable catechol 2,3-dioxygenase

Catechol 2,3-dioxygenase (C23O) in one of extradioltype dioxygenases which cleave the aromatic C-C bond at meta position of the hazardous dihydroxylated aromatic substrates, and it plays a key role in the degradation of aromatic molecules by soil bacteria in the environment[1,2]. In a high temperature environment, the counterpart of the mesophilic C23O is the thermostable catechol 2,3-dioxygenase (TC23O) from thermophiles. The pheB gene encoding TC23O from the thermophile Bacillus stearothermophilus FDTP-3 was cloned[3] and subsequently subcloned into the plasmid pJLA503, and it was over expressed in E.coli TG 1 and crystallized by our lab[4,5]. Analytical results reveal that TC23O is a homotetramer with an apparent molecular mass of 140 kD, and each subunit is composed of 327 amino acid residues. The homology in amino acid sequence between the mesophilic C23O (from Alcaligenes eutrophus 335[2]) and the thermophilic TC23O is about 27%.

TC23O is an ideal model protein for exploring the mechanism and structural basis for protein thermostability owing to the ease and high sensitivity in its assay. In order to investigate the effects of amino acid replacement on the thermostability of TC23O, random PCR mutagenesis was utilized to generate various mutants with changed thermostability. Pro229Ser is one of these mutants, the thermostability of which is decreased compared with that of the wild type TC23O, which implies that Pro229 is probably important for the thermostability of TC23O. As a preliminary step in the study of the molecular basis of thermostability, we report here the expression, purification, crystallization and preliminary X-ray analysis of Pro229Ser.

1    Materials and Methods

1.1  Expression and purification

E.coli TG 1, transformed with the wild type or mutant pheB gene, was grown in 2×YT medium (containing 100 mg/L ampicillin) at 30 ℃ until A600 reached 0.8-1.0, and protein expression was induced for about 8 h by shifting the temperature to 42 ℃. The cells were harvested by centrifugation and were lysed by sonication in buffer A (containing 20 mmol/L Na2HPO4-NaH2PO4 at pH 8.0, 2 mmol/L EDTA, and 10 mmol/L b-mercaptoethanol). Crude extracts were applied to a DEAE-Sepharose Fast Flow column (Pharmacia) pre-equilibrated with buffer A. This column was washed with buffer A and then eluted with a linear gradient of 0-1 mol/L NaCl in buffer A. The eluted fractions containing TC23O were pooled and dialyzed against buffer B(containing 20 mmol/L Na2HPO4-NaH2PO4 at pH 8.0, 0.8 mol/L (NH4)2SO4, 2 mmol/L EDTA, and 10 mmol/L b-mercaptoethanol) for 8 h at 4 ℃ and then loaded onto a Phenyl Sepharose 6 Fast Flow column (Pharmacia) pre-equilibrated with buffer B. This column was washed with buffer B and eluted with a linear (NH4)2SO4 gradient from 0.8 mol/L to 0 mol/L in buffer A. Enzyme purity was evaluated by 12% SDS-PAGE, and protein concentration was determined by the Bradford method[6].

1.2  Analytical methods

Assay of TC23O activity was carried out in buffer A at 60 ℃ with catechol as a substrate by monitoring the absorbance increase at 375 nm (A375) due to the formation of the product a-hydroxymuconic e-semialdehyde[1]. Kinetic parameters were estimated from the intercepts of Lineweaver-Burk plots. The thermostability of wild type TC23O or the mutant Pro229Ser was evaluated by incubating each enzyme solution at different temperature from 25 ℃ to 100 ℃ (with an interval of 5 ℃) for 15 min, respectively, and cooling down immediately in an ice bath, and then assaying the residual enzyme activity at 60 ℃ as described above. The temperature (Tm) corresponding to 50% residual enzyme activity was used to estimate the thermostability of the enzyme.

1.3  Crystallization and X-ray diffraction analysis

The purified enzyme (wild type TC23O or its mutant Pro229Ser) in buffer A was placed in a ultrafiltration concentrating tube (Millipore) fitted with a 30 kD cut-off membrane and the sample was centrifuged according to the manufacture's instructions. When the volume in the concentrating tube had fallen to approximately 100 ml , 1 ml of 0.1 mol/L HEPES buffer (pH 7.5) was added. This treatment was repeated at least 8 times. At the end of this procedure, the concentration of the purified enzyme was approximately 20 g/L determined by the Bradford method[6]. Crystals were grown by using the hanging-drop vapor-diffusion method in Costar 24-well plate at 4 ℃. A number of different crystallization conditions were screened. The optimal crystallization solution consisted of 33% PEG 400, 0.2 mol/L MgCl2, and 0.1 mol/L HEPES buffer (pH 7.5). The reservoir contained 700 ml of this solution. The hanging drop contained 5 ml of the concentrated protein and 5 ml of the crystallization solution.

X-ray diffraction data were collected at room temperature on a MarResearch Imaging Plate (diameter 300 mm) at the Young Scientist Laboratory of Structure Biology, University of Science and Technology of China (USTC) in Hefei. Cu Ka radiation was generated at 40 kV and 50 mA. The crystal-to-detector distance was set at 175 mm, and 100 images were recorded at 1° interval. The exposure time was 600 s per image. DENZO[7] was used to determine the unit-cell parameters and space group. All data were indexed, integrated, scaled and reduced with DENZO and SCALEPACK programs on a Silicon Graphics INDY system.

2    Results and Discussion

The mutant and wild type proteins were expressed and purified as described above. The amount of expression was about 30% in total bacterial proteins, and the yields of proteins from 1 L 2×YT medium were about 15-20 mg. The purified proteins proved to be homogeneous by SDS-PAGE (data not shown). The kinetic and thermostability parameters of the mutant Pro229Ser and wild type TC23O were shown in Table 1.

 

Crystals of the mutant Pro229Ser, suitable for X-ray analysis, were grown to approximately 0.6 mm×0.5 mm×0.3 mm within one week under the optimal conditions as described above (Fig.1). X-ray diffraction data collection statistics are summarized in Table 2. The crystals diffracted to at least 0.24 nm resolution. A total of 110 786 observed reflections were scaled and reduced to yield a data set containing 14 156 unique reflections. In the 0.24-10.0 nm resolution range, the overall completeness was 92.4%, Rmerge was 11.9%, and I/s(I) was 10.5. The space group of these crystals was determined to be orthorhombic I222 with unit-cell parameters a＝7.059 nm, b＝7.415 nm, c＝13.311 nm. Assuming the presence of 2 molecules of the mutant enzyme Pro229Ser in the asymmetric unit, the Matthews parameter (Vm) was calculated to be 2.49×10-3 nm3·D–1, and the solvent content was about 51% of the unit-cell volume[8].

 

Fig.1  Crystal of the mutant enzyme Pro229Ser

 

 

Many studies on the comparison of different thermophilic proteins and/or their mutants suggested that amino acid substitutions might cause the adjustments of the local conformation in the vicinity of the altered amino acid owing to the changes of the volume, hydrophobicity, and polar properties of the residue. These kinds of local conformational adjustments altered the properties of the specific proteins (such as enzymatic characters, thermostability, etc.)[9-12]. Our results agreed with these viewpoints. The characteristic analysis (Table 1) revealed that the kinetic parameters of the enzyme were slightly different between the wild type TC23O and its mutant Pro229Ser, however, the thermostability of Pro229Ser was decreased, the Tm being about 10.2 ℃ lower than that of the wild type TC23O, which implies that Pro229 might play an important role in the thermostability of TC23O.

It is obvious that crystallographic three-dimensional structure studies of TC23O and its mutants would not only lead to an explanation of the effects of amino acid replacements on the thermostability of TC23O, but also lead to an insight into the mechanism and structure basis for the thermostability of TC23O. The crystal structure determination of the wild type TC23O and its mutant enzyme Pro229Ser is currently under way.

 

Acknowledgments    We thank Professor NIU Li-Wen, Professor TENG Mai-Kun and Professor GONG Wei-Min at the Young Scientist Laboratory of Structure Biology, USTC, for their kindly help in X-ray data collection and analysis. We also thank Professor ZHANG Zhi-Hong and Professor CHENG Min-Qing of Fudan University for their help and advice in this work.

 

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Received:April 11, 2001  Accepted:May 29, 2001

This work was partly supported by the National Natural Science Foundation of China (No.39870402, No.30070161)

*Corresponding author: Tel, 86-21-65643958; Fax, 86-21-65642502; e-mail, [email protected]