crystallization communications
216
doi:10.1107/S174430910900092X Acta Cryst. (2009). F65, 216–218Acta Crystallographica Section F Structural Biology and Crystallization Communications ISSN 1744-3091
Purification, crystallization and preliminary X-ray
analysis of an aminoacylhistidine dipeptidase
(PepD) from
Vibrio alginolyticus
Chin-Yuan Chang,aYin-Cheng Hsieh,b,cTing-Yi Wang,a Chun-Jung Chenb,d,e* and Tung-Kung Wua*
aDepartment of Biological Science and
Technology, National Chiao Tung University, Hsinchu 30010, Taiwan,bLife Science Group,
Scientific Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan,cInstitute of
Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan,
dDepartment of Physics, National Tsing Hua
University, Hsinchu 30013, Taiwan, and
eInstitute of Biotechnology, National Cheng
Kung University, Tainan 701, Taiwan
Correspondence e-mail: cjchen@nsrrc.org.tw, tkwmll@mail.nctu.edu.tw
Received 1 November 2008 Accepted 8 January 2009
The aminoacylhistidine dipeptidase (PepD) protein encoded by Vibrio algino-lyticus pepD was successfully overexpressed and characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The purified enzyme contained two zinc ions per monomer. The recombinant dipeptidase enzyme, which was identified as a homodimer in solution, exhibited broad substrate specificity for Xaa-His dipeptides, with highest activity towards the His-His dipeptide. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Preliminary crystallographic analysis showed that the crystal belonged to space group P61or P65, with unit-cell parameters
a = b = 80.42, c = 303.11 A˚ . The crystal contained two molecules per asymmetric unit and the predicted solvent content was 53.4%.
1. Introduction
Vibrio alginolyticus is one of the most common and important pathogens that cause vibriosis in human and marine species (Morris & Black, 1985; Levine & Griffin, 1993; Marano et al., 2000). For example, infection of fish by V. alginolyticus results in biofilm formation in their intestine, which leads to fish mortality and potentially significant economic losses (Lee, 1995). Human infection by V. alginolyticus is caused by the consumption of raw or under-cooked seafood (Blake et al., 1980; Rose et al., 2001). The major clinical symptoms of V. alginolyticus infection of humans are wound infection, gastroenteritis and septicaemia (Blake et al., 1980; Hlady & Klontz, 1996; Rose et al., 2001). Thus, prevention, early detection and treatment of V. alginolyticus infections are important for the health of humans and marine animals.
Aminoacylhistidine dipeptidase (PepD; EC 3.4.13.3) is a member of metallopeptidase family M20 in the metallopeptidase H (MH) clan (Rawlings & Barrett, 1995). The majority of cocatalytic
metallo-hydrolases are Zn2+-dependent enzymes, but some require Mn2+,
Co2+ or other divalent metal ions for activity. The PepD enzyme
catalyzes the cleavage and release of an N-terminal amino acid (usually a neutral or hydrophobic residue) from an Xaa-His dipeptide or other degraded peptide fragments (Rawlings & Barrett, 1995). The enzyme also exhibits a broad substrate specificity which includes the unusual dipeptides carnosine (-Ala-His) and homocarnosine (-amino-butyl-His) and several distinct tripeptides. These enzymes play fundamental roles in certain biochemical events, such as protein maturation and degradation, tissue repair and cell-cycle control (Chen et al., 2008). The enzymes of the M20 family may also play fundamental roles in aging and neurodegenerative or psychiatric diseases (Teufel et al., 2003) and hence may have great potential for application as antibacterial targets or therapeutic agents (Hellen-doorn et al., 1997).
The pepD gene from V. alginolyticus contains an open reading frame (ORF) of 1473 nucleotides and codes for a 490-amino-acid protein. Bioinformatic analysis of the V. alginolyticus PepD sequence reveals its high homology to those from other Vibrio species (94–76% identity) and bacteria (75–63% identity). Sequence-based alignment
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of V. alginolyticus PepD with peptidase clan MH proteins of known structure, such as Aeromonas (Vibrio) proteolyticus aminopeptidase (AAP; Chevrier et al., 1994), Streptomyces griseus aminopeptidase S (SGAP; Greenblatt et al., 1997), Pseudomonas sp. carboxypeptidase G2 (CPG2; Rowsell et al., 1997), Salmonella typhimurium PepT (Hakansson & Miller, 2002), human aminoacylase 1 (hACy1; Lindner et al., 2003) and Lactobacillus delbrueckii peptidase V (PepV; Jozic et al., 2002), showed low sequence identities in the range 7–20% and low sequence similarities in the range 13–34%. Despite the lack of detectable sequence homology, the putative active-site residues for catalysis are relatively conserved in PepD and related di-zinc enzymes from the M20 family (Rowsell et al., 1997; Jozic et al., 2002). His80, Asp119, Glu150, Asp173 and His461 have been predicted to be involved in metal binding, whereas Asp82 and Glu149 are essential for catalysis. These residues were completely conserved apart from Asp173, which is present in homologues with amino-peptidase/dipeptidase specificity, whereas aminoacylase/carboxy-peptidases contain a glutamic acid at the same position. Here, we report the cloning and expression of the V. alginolyticus pepD gene, the purification of the produced recombinant PepD protein and the preliminary X-ray diffraction characterization prior to crystal struc-ture determination to reveal the overall structural feastruc-tures and the spatial locations of the putative active-site residues.
2. Materials and methods
2.1. Molecular cloning of theV. alginolyticus pepD gene
The V. alginolyticus pepD gene was amplified from the V. algino-lyticus ATCC 17749 genomic DNA library. Two primers (F1,
50-GTGTCTGAGTTCCATTC-30, and R1, 50
-TTACGCCTTTTCA-GGAA-30), based on the highly conserved 50-end and 30-end nucleic acid sequences of Vibrio spp. pepD, were designed to amplify the putative V. alginolyticus pepD gene. An ORF that contains 1473 nucleotides and codes for a polypeptide chain of 490 amino acids was identified. Sequence analysis indicated a protein with an estimated molecular mass of approximately 53.6 kDa and an isoelectric point of pH 4.7. The nucleotide sequence of the V. alginolyticus pepD gene was deposited in the GenBank database (accession No. DQ335448).
2.2. Production and purification of recombinantV. alginolyticus PepD protein
The V. alginolyticus pepD gene was subcloned into the expression plasmid pET28a(+) and subsequently transformed into Escherichia coli BL21(DE3)pLysS cells for recombinant protein production and purification. The production of PepD protein was induced by the addition of 0.5 mM isopropyl -d-1-thiogalactopyranoside (IPTG) and continuous incubation for 6 h at 310 K. After 6 h incubation with rotary shaking at 310 K, the cells were collected by centrifugation at 9300g for 30 min at 277 K. The medium was discarded and the cell pellet was resuspended in binding buffer (10 ml) containing 0.5 M NaCl and 20 mM Tris–HCl pH 6.8. The resuspended cells were disrupted by sonication using a 2 s on/1 s off pulsation cycle with a total sonication time of 3 min at 30% energy on ice. The supernatant was collected by centrifugation at 12 000g for 30 min at 277 K. The recombinant PepD protein was purified using an Ni–NTA column and eluted with imidazole. The crude protein containing PepD was loaded onto an Ni–NTA column that had previously been washed with a ten-column volume of buffer A (0.5 M NaCl, 20 mM Tris–HCl pH 6.8) and 60 mM imidazole. The PepD protein was eluted with buffer A containing stepwise increasing concentrations of 100, 200, 300 and 500 mM imidazole. The eluted fractions with PepD enzy-matic activity were collected and dialyzed into 50 mM Tris–HCl pH 6.8 before use in crystallization. The purified PepD was concentrated
to 10 mg ml1using a Centricon (10 kDa molecular-weight cutoff;
Amicon Ultra, Millipore) in 20 mM Na HEPES buffer pH 6.8. The purity of the protein was confirmed using 12.5% SDS–PAGE with Coomassie Brilliant Blue R-250 staining.
2.3. Crystallization
Crystallization trials were carried out using the hanging-drop vapour-diffusion method at 291 K and initial screening of crystal-lization conditions was carried out using Crystal Screens 1 and 2 (Hampton Research) in 48-well plates at 293 K. 1 ml protein solution (10 mg ml1) and 1 ml reservoir solution were mixed in each drop and the drops were equilibrated against 200 ml reservoir solution.
2.4. X-ray data collection and processing
The protein crystals were initially screened and characterized at the National Synchrotron Radiation Research Center (NSRRC) in Taiwan using synchrotron-radiation X-rays on the SPXF beamline BL13B1 equipped with a CCD (Q315, ADSC) detector. Data
crystallization communications
Acta Cryst. (2009). F65, 216–218 Chang et al. PepD
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Figure 1
12.5% SDS–PAGE of the purified V. alginolyticus PepD protein. Lane M, marker proteins (kDa): phosphorylase b (97 kDa), bovine serum albumin (67 kDa), ovalbumin (45 kDa) and carbonic anhydrase (30 kDa). Lane 1, crude cell extracts of E. coli BL21(DE3)pLysS carrying the pET-28a(+) plasmid. Lane 2, crude cell extracts of E. coli BL21(DE3)pLysS carrying the pET-28a(+)-pepD plasmid. Lane 3, purified PepD from the Ni–NTA column.
Figure 2
collection was completed at the Taiwan-contracted beamline BL12B2 equipped with a CCD (Quantum-4R, ADSC) detector at SPring-8 in Japan. The crystal was transferred into a cryoprotectant solution containing 15%(v/v) glycerol for a few seconds, mounted on a loop (0.2–0.3 mm; Hampton Research) and then flash-cooled in liquid
nitrogen. For complete X-ray data collection, 300 of rotation were
measured in 1.0oscillations using X-rays of wavelength 1.00 A˚ with 15 s exposures and a crystal-to-detector distance of 350 mm at 110 K in a nitrogen stream generated using a cryosystem (X-Stream, Rigaku Inc.). All data were indexed, integrated, scaled and merged using HKL-2000 (Otwinowski & Minor, 1997).
3. Results and discussion
The putative aminoacylhistidine dipeptidase PepD from V. algino-lyticus was overproduced in E. coli and purified. SDS–PAGE of the purified PepD showed a single band with a molecular mass of approximately 54 kDa (Fig. 1). Sequence and structural homologies suggested that the enzyme was a member of the metal-dependent metallopeptidase family. Homology comparisons of V. alginolyticus PepD, using L. delbrueckii PepV as the template, revealed that the putative metal-binding residues are almost superimposed on each other, with the exception of Asp119, which is likely to be the residue that holds the two zinc ions in PepV (Jozic et al., 2002). The conserved Asp119 of PepD aligned with Asp120 of PepV. This residue is adja-cent to the metal-binding residue Asp119, whereas Ala118 in PepD is located at the corresponding position to Asp119 in PepV (Wang et al., 2008).
Good-sized diamond-shaped crystals appeared within six months in a condition consisting of 100 mM Na HEPES buffer pH 7.5,
28%(v/v) PEG 400 and 200 mM CaCl2 and grew to maximum
dimensions of 0.3 0.2 0.1 mm (Fig. 2). The protein crystals were sensitive to the change in precipitant concentration on transfer to the cryoprotectant solution containing 15%(v/v) glycerol. Good-quality crystals were carefully screened and selected for data collection as
they frequently possessed fairly high mosaicities (>1). The crystals produced diffraction data to 3.0 A˚ resolution on beamline BL12B2 at SPring-8. Analysis of the diffraction pattern revealed that the crystals exhibited hexagonal symmetry and systematic absences indicated the space group to be P61or P65. The unit-cell parameters were a = 80.42,
c = 303.11 A˚ . If we assume the presence of two molecules in the
asymmetric unit and a molecular mass of 54 kDa, the calculated
solvent content is 53.4% and the Matthews coefficient (VM) is
2.63 A˚3Da1(Matthews, 1968), which is within the normal range for protein crystals. The statistics of the collected data are summarized in Table 1. Structure determination by the molecular-replacement method using the aminopeptidase PepV from L. delbrueckii (Jozic et al., 2002) as a search model is currently under way.
We thank the National Chiao Tung University, the MOE ATU program and the National Science Council (NSC-96-2627-M-009-003) for their financial support of this research. This study was also supported by grants from the National Synchrotron Radiation Research Center (963RSB02) and the National Science Council (NSC 95-2321-B-213-001-MY3) in Taiwan to C-JC. We are grateful to our colleagues Yuch-Cheng Jean and Chun-Shiun Chao and the support staff for technical assistance during data collection on BL13B1 of NSRRC in Taiwan and to Jeyaraman Jeyakanthan at BL12B2 of SPring-8 in Japan.
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Chang et al. PepD Acta Cryst. (2009). F65, 216–218Table 1
X-ray diffraction statistics for the PepD crystal.
Values in parentheses are for the highest resolution shell.
Wavelength (A˚ ) 1.00 (SPring-8 BL12B2) Temperature (K) 110 Resolution range (A˚ ) 30.0–3.0 Space group P65 Unique reflections 54648 Completeness (%) 99.8 (99.9) hI/(I)i 23.8 (6.2) Average redundancy 4.3 Rmerge† (%) 6.1 (32.3) Mosaicity () 0.6 Unit-cell parameters (A˚ ) a = 80.42, c = 303.11
No. of molecules per ASU 2
VM(A˚3Da1) 2.63 Solvent content (%) 53.4 † Rmerge= P hkl P ijIiðhklÞ hIðhklÞij= P hkl P
iIiðhklÞ, where Ii(hkl) is the ith
observa-tion of reflecobserva-tion hkl and hI(hkl)i is the weighted average intensity for all observaobserva-tions i of reflection hkl.
