1 Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan
2 Faculty of Nutrition, Kobegakuin University, Kobe, Hyogo 651-2180, Japan
3 Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680-8552, Japan
Correspondence
Fusako Kawai
fkawai{at}rib.okayama-u.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AB190288.
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INTRODUCTION |
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In this paper, we report the purification and characterization of OPH from Sphingomonas sp. strain 113P3 (formerly Pseudomonas sp. 113P3). Cloning and sequencing of the gene was also performed. The results show that OPH from strain 113P3 has certain unique characteristics such as its localization, substrate specificities and molecular mass, as well as the gene itself.
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METHODS |
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Bacterial strains and cultivation.
Sphingomonas sp. strain 113P3 (formerly identified as Pseudomonas sp. 113P3) was used throughout. The strain has the accession number FERM P-13483 in the International Patent Organism Depositary (IPOD) (Tsukuba, Japan). The strain was grown on PVA medium (pH 7·5) as reported previously (Hatanaka et al., 1995a). The glucose medium contained the same components as PVA medium except that glucose was added instead of PVA117. The bacterium was also grown on nutrient broth (Eiken Chemical Co.). The cells were harvested by centrifugation, washed twice with 0·85 % NaCl and kept at 80 °C until use. E. coli DH5
and E. coli transformants were grown at 37 °C in LB medium, supplemented with 50 µg ampicillin ml1 when necessary.
Taxonomic identification.
As genetic evidence for identification, the partial 16S rDNA (rDNA) sequence of strain 113P3 was analysed, based on the methods of Rochelle et al. (1995). Nucleotide sequencing was carried out using an ABI PRISM 377-18 DNA sequencer and a BigDye Terminator Cycle sequencing Kit (Applied Biosystems) according to the manufacturers' instruction manuals.
Enzyme assay.
Preliminary studies on the substrate specificity of the purified enzyme revealed that OPH acted on oxidized PVA and p-nitrophenyl acetate (PNPA). Therefore OPH activity was routinely measured by using PNPA as a substrate. The reaction mixture contained 1 mM PNPA and 50 mM potassium citrate buffer (pH 6·0), which was preincubated at 37 °C for 1 min. The reaction was started by the addition of enzyme solution and carried out at 37 °C for 20 min. The enzyme activity was assayed in duplicate by measuring the A415 (due to p-nitrophenol liberated; =2591 M1 cm1) with a Shimadzu UV-160 spectrophotometer (1 cm light path). A reaction mixture without enzyme solution was used as a reference. One unit of enzyme activity was defined as the amount of enzyme that catalysed the hydrolysis of 1 µmol PNPA min1 under the assay conditions. Activity toward oxidized PVA was measured by the decrease of A300 in carbonate buffer (pH 10·0:
=14·6 mM1 cm1), as described previously (Shimao et al., 2000
). The calculation for oxidized PVA was done as follows. As the oxidation rate of hydroxyl groups in PVA 500 was 4 %, 10 mol diketone structures must exist among 500 hydroxyl groups in 1 mol PVA 500. Thus hydrolysis of 1 mol diketone corresponded to 0·1 mol oxidized PVA. Based on this calculation, specific activity and Km and Vmax values were measured. The activity toward mono- and diketones and esters was assayed by decrease in pH and determination of the carboxylic acid formed, which was spectrophotometrically analysed according to the method of Kasai et al. (1975)
, measuring the colour development with ferric hydroxamate of carboxylic acids. PVADH activity was measured as described previously (Shimao et al., 1986
).
Preparation of cell-free extracts, and periplasmic, cytoplasmic and membrane fractions.
Cells grown on PVA were suspended in 50 mM Tris/HCl buffer (pH 8·0) and sonicated with a UD-200 ultrasonic disruptor (Tomy Seiko Co.) at 20 kHz for 10 min below 4 °C, and the sonicate was centrifuged at 16 000 g for 30 min to remove unbroken cells and cell debris. The resultant supernatant was used as cell-free extract. The method of Anraku & Heppel (1967) was modified for preparation of the periplasmic, cytoplasmic and membrane fractions. The cell paste (wet weight 190 g obtained from 5 l culture on PVA medium) was suspended in an appropriate amount of 30 mM Tris/HCl buffer (pH 8·0) containing 30 % (w/v) sucrose and 1 mM EDTA, and gently stirred at room temperature for 15 min to cause plasmolysis. The mixture was centrifuged at 16 000 g for 30 min at 4 °C to obtain the plasmolysed cells. The supernatant was used as the sucrose-EDTA fraction. The plasmolysed cells were suspended in 10 vols cold water containing 1 mM MgCl2, and the suspension was gently stirred for 30 min on ice. The mixture was centrifuged at 16 000 g for 30 min at 4 °C to remove the osmotically shocked cells, and the supernatant was used as the cold-water fraction. The sucrose-EDTA and the cold-water fractions were combined and used as the periplasmic extract. The osmolysed cells were sonicated with a UD-200 ultrasonic disruptor at 20 kHz for 10 min below 4 °C and centrifuged at 68 000 g for 90 min at 4 °C. The supernatant and pellets were used as the cytoplasmic and membrane fractions, respectively.
Activities of glucose-6-phosphate dehydrogenase (Bergmeyer et al., 1974) and alkaline phosphatase (Garden & Levinthal, 1960
) were also measured as cytosolic and periplasmic marker enzymes, respectively.
Purification of OPH.
The periplasmic extract was dialysed against 10 mM Tris/HCl buffer (pH 8·5) at 4 °C overnight, with several changes of the buffer. The pellets formed during dialysis were discarded by centrifugation. The supernatant was applied to a DEAE-Sepharose column (2·5x10 cm) pre-equilibrated with 20 mM Tris/HCl buffer (pH 8·5). The column was washed with two bed volumes of the same buffer and the elution was performed with a linear gradient from 0 to 0·5 M NaCl in the same buffer. The activity was found only in the unbound fractions, which were collected and concentrated by ultrafiltration with a YM 30 Diaflo membrane (Millipore). PVADH activity was eluted from the bound fractions and used for preparation of oxidized PVA. The concentrated enzyme was dialysed against 10 mM acetate buffer (pH 6·0) overnight and applied to a CM-Sepharose column (1·8x6 cm), pre-equilibrated with the dialysis buffer. The column was washed with two bed volumes of the buffer and eluted with a linear gradient from 0 to 0·3 M NaCl in the same buffer. The active fractions were pooled and ammonium sulfate was added to 0·3 M. Then the enzyme solution was applied to a Phenyl Sepharose column (2·5x10 cm) pre-equilibrated with 50 mM Tris/HCl buffer (pH 7·6) containing 0·3 M ammonium sulfate. The column was washed with two bed volumes of the buffer and eluted with a linear gradient from 0·3 to 0 M ammonium sulfate. The active fractions were pooled and used as the purified enzyme preparation. The purified enzyme was stable at 4 °C for several weeks.
Analyses.
The protein concentration was determined by a Bio-Rad Protein Assay kit with bovine serum albumin as the standard. The homogeneity of the protein and the molecular mass of the enzyme subunit were confirmed by SDS-PAGE based on the method of Laemmli (1970). The molecular mass of the native OPH was determined by a SMART System (Amersham Pharmacia) on a Superdex 200 column (3·2x300 mm) equilibrated with 50 mM Tris/HCl buffer (pH 7·5) containing 100 mM NaCl and 1 mM MgCl2, with phosphorylase b (94 kDa); 2, bovine serum albumin (67 kDa); 3, ovalbumin (43 kDa) as size standards. The N-terminal amino acid sequence of the purified enzyme was determined with a Procise 491 protein sequencer (Applied Biosystems). Internal amino acid sequences were analysed by the method of Aebersold et al. (1987)
. Homology searches were performed with the BLAST program (http://blast.genome.ad.jp/).
The Mn of oxidized PVA was measured by HPLC, performed with a Tosoh CCPM-II liquid chromatograph. The analytical conditions were as follows: detection, Tosoh RI-8020; columns, Tosoh TSK-GEL2500PW connected with TSK-GEL3000PW; eluent, 0·3 M NaNO3; flow rate, 1 ml min1; column temperature, 40 °C. Molecular masses were measured using ethylene glycol, its oligomers and polyethylene glycols, and TSK standard polyethylene oxides (Tosoh).
Cloning of the OPH-encoding gene (oph).
DNA purification, transformation and electrophoresis were performed as described by Sambrook & Russell (2001). Ex Taq DNA polymerase was routinely used for PCR under the conditions recommended by the manufacturer (Takara Bio Co.). The PCR products were sequenced for both strands. To prepare a probe DNA for screening the oph gene, 5'- and 3'- degenerate primers were designed based on the N-terminal and internal amino acid sequences of the purified enzyme, followed by nested PCR (Olsvik et al., 1991
) to amplify the specific fragment. In the first PCR, the primers used were Nt1 [5'-GA(G/A)TGGGC(G/A/C)TGCCC(G/A/C/T)GA(G/A)GG-3'] and In1 [5'-CC(C/T)TT(G/A)TA(G/A)TG(G/A)TC(C/T/G)GT(G/A)AA-3']. For the nested PCR, 1 µl of the first PCR reaction mixture was used and the primers Nt2 [5'-TGGGC(G/A/C)TGCCC(G/A/C/T)GA(G/A)GG(A/C)TTC-3'] and In2 [5'-GG (G/A/C)AC(C/T/G)GT(G/A/C/T)GA(T/G)CC(G/A)TC(G/A)TC-3'] were used for the reaction. The product of 500 bp was purified and ligated into a pGEM-T easy vector (Promega). The plasmid (pOPH-p) was transformed and extracted from transformant E. coli.
Inverse PCR (Ochman et al., 1988) was performed to amplify the region surrounding oph. NaeI-digested and self-ligated chromosomal DNA was used as a template and amplified with the primer pair OPH-Inv-F (5'-GACCATCGGAACCCACACGG-3') and OPH-Inv-R (5'-GCCGGTGGAATGCCGATCTC-3'). The amplified 2·5 kb DNA fragment was ligated into a pGEM-T easy vector (pOPH-i) and sequenced. In the downstream region of oph, a gene encoding PVADH was found, which was already deposited in GenBank database under accession no. D83772. Further downstream region (1 kb EcoRI fragment) was cloned into pBluescript II SK+ (Stratagene) by the colony hybridization method (Sambrook & Russell, 2001
). The sequence including oph, the PVADH-encoding gene (pvaA) and the putative cytochrome c gene (cytC) was deposited in GenBank under accession no. AB190288.
Construction of the expression vector for oph.
The ORF of oph except for the putative signal peptide region was amplified by PCR using the primers ExOPH-N (5'-GAGCTCTAAGGAGGTTTTTATATGAAGGCGAATGGGCCTGCCCCG-3'), and ExOPH-C (5'-AAGCTTTCATTTGTAATGATC-3'), which contained SacI and HindIII sites (underlined) and the ShineDalgarno sequence (italicized). The amplified fragment was first ligated into a pGEM-Teasy vector and cut by SacI and HindIII, and then ligated into the corresponding position of a pUC118 vector. The resultant plasmid (pUC-oph) was transformed into E. coli DH5. The transformants were grown on LB medium at 37 °C for 2 h and the expression of oph was induced by the addition of 1·0 mM IPTG.
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RESULTS |
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Inductivity and localization of OPH and PVADH
The inductivity of OPH and PVADH was studied with cells grown on PVA medium, glucose medium and a nutrient broth, and the effect of the addition of PQQ was studied as well. The organism could not grow on PVA in the absence of PQQ. OPH activity was detected in cell extracts obtained after growth on different media. ApoPVADH was expressed in all the media, but its activity was detected only if PQQ was added. Thus both OPH and PVADH were constitutively formed. Since the best medium was PVA medium supplemented with PQQ, the following experiments were performed with cells grown on this medium. Most of the PVADH and OPH activities were present in the periplasmic fraction (Table 1). Shimao et al. (2000)
provided evidence that PVADH and OPH are membrane-bound enzymes in Pseudomonas sp. VM15C. Therefore, we conclude that the reaction sites of PVA degradation in Sphingomonas sp. strain 113P3 and in Pseudomonas sp. VM15C are in the periplasm.
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Km and Vmax values for oxidized PVA and PNPA were 0·2 and 0·3 mM, and 0·1 and 3·4 µmol min1 mg1, respectively. Whether the OPH was really active toward oxidized PVA was confirmed by decrease in A300 (for diketone structure) (Silverstein et al., 1991; Shimao et al., 2000
) and the shift of Mn of oxidized PVA. An increase in A300 up to 60 min due to PVADH activity and then a decrease due to OPH activity were found (Fig. 1a, b
). Both reactions were dependent on the amounts of the enzymes, suggesting that these enzymes catalysed both reactions. Oxidized PVA was hydrolysed by OPH, which was analysed by HPLC (Fig. 1c
). Oxidized PVA prepared by PVADH showed two peaks on HPLC, corresponding to Mn values of approximately 11 000 (47 %) and 1400 (53 %). The former peak was shifted to Mn values ranging from 7500 to 3700 and the latter peak increased in height after hydrolysis by OPH.
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Cloning and sequencing of the oph gene
Based on the N-terminal and internal amino acid sequences, nested PCR with degenerate primers was performed and a fragment of 500 bp was amplified (pOPH-p). The region surrounding oph was amplified by inverse PCR. The 2·5 kb fragment was obtained and cloned into a cloning vector (pOPH-i). Further downstream region was cloned by the colony hybridization method (pOPH-c). Joining the three plasmid sequences (pOPH-p, pOPH-i and pOPH-c) resulted in a DNA sequence (3827 bp) containing three open reading frames, encoding OPH (oph), PVADH (pvaA) and a putative cytochrome c (cytC) (Fig. 2). The ORF of oph consists of 1095 bp, corresponding to a protein of 364 amino acid residues, encoding a signal peptide and a mature protein of 34 and 330 amino acid residues, respectively. The deduced amino acid sequence was in accordance with the N-terminal and internal amino acid sequences of the purified OPH. The presence of the serine-hydrolase motif (lipase box, Gly-X-Ser-X-Gly) strongly suggests that the oph-encoded protein belongs to the serine-hydrolase family (Pelletier et al., 1995
). The putative amino acid sequence of oph exhibited homology to OPH from Pseudomonas sp. strain VM15C (63 % identity), and to the polyhydroxyalkanoate (PHB) depolymerases from Mesorhizobium loti, Rhizobium sp. and Sinorhizobium meliloti strain 1021 (2932 % identity).
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In the downstream region of oph, a gene encoding PVADH (pvaA) was located. The putative amino acid sequence of the gene exhibited homology with PVADH from strain VM15C (52 % identity). Furthermore, a putative cytochrome c gene located downstream of pvaA was sequenced.
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DISCUSSION |
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The OPH from Sphingomonas sp. strain 113P3 was purified for the first time and characterized. PVADH and OPH are constitutively produced, as is already known to be the case for other PVADHs (Matsumura et al., 1998, 1999
). The inductivity of OPHs from strain VM15C and Pseudomonas vesicularis PD (Sakai et al., 1985
, 1986
) has not been documented. The molecular masses of the native enzyme and a monomer protein were estimated to be 70 kDa and 35 kDa, respectively, indicating that the enzyme is a homodimer. The gene for the OPH of Pseudomonas sp. strain VM15C was cloned, but since the enzyme was not purified, its enzymic characteristics are not clear (Shimao et al., 2000
). The enzyme of strain 113P3 was inhibited by PMSF in a manner similar to that of strain VM15C, suggesting that the active sites of both enzymes include serine residues (Saegusa et al., 2002
; Zhang et al., 1997
). The enzyme hydrolysed PNPA and oxidized PVA, but did not hydrolyse any mono- or diketones. Shimao et al. (2000)
reported that the cell extract of E. coli harbouring oph has an activity toward oxidized PVA about 350 times greater than that toward 4,6-nonanedione (diketone). Thus, the OPH from strain 113P3 exhibits a different specificity. Shimao et al. (2000)
measured OPH activity toward oxidized PVA only by A300, but did not confirm whether the polymer was depolymerized. In this paper, the Mn (11 000) of oxidized PVA was clearly shifted to lower Mn values (75003700) and a peak at Mn 1400 increased, showing that the polymer was actually depolymerized. Mn values of oxidized PVA (11 000 and 1400) formed from PVA 500 (Mn 22 000) might be due to non-enzymic cleavage during enzymic oxidation and concentration after reaction or contaminating esterase activity.
We cloned the gene for OPH and found that the open reading frame consists of 1095 bp, corresponding to 364 deduced amino acid residues, encoding a signal peptide and a mature protein of 34 and 330 residues, respectively. By BLAST analysis, OPH exhibited a high homology (63 % identity) to the OPH from Pseudomonas sp. VM15C, and to PHB depolymerases from various sources, but no significant homology to other enzymes including lipases/esterases. The amino acid sequences of the OPHs from strain 113P3 and strain VM15C were aligned by CLUSTAL W (Thompson et al., 1994) with the hypothetical PHB depolymerases from Mesorhizobium loti, Rhizobium sp. and Sinorhizobium meliloti strain 1021, to which the OPH from strain 113P3 showed homology (2932 % identity) and the well-characterized PHB depolymerases from Pseudomonas lemoignei (PhaZ1 to PhaZ5; Jendrossek et al., 1995b
) (Fig. 3
). PHB depolymerases share common structural domains conserved in the group as a whole: in the N-terminus, the signal peptide, the catalytic domain including the lipase box, the threonine-rich region or the type III module of fibronectin, and the substrate-binding site (Jendrossek et al., 1995a
, b
). The primary structure of the catalytic domain of these depolymerases contains certain conserved structures such as an oxyanion hole (histidine) and a triad of three amino acid residues (serine, aspartate and histidine) that is conserved among the serine proteases (Brenner, 1988
; Kim et al., 2004
; Lassy & Miller, 2000
). Their consensus sequences are L***lHGC-QtAs, ID-n-vYV-GLS-G+--t, vw-G-sDyTV, and GM-H--P---G, respectively (* indicates hydrophobic, + a small side chain, and the corresponding residues are underlined). These structures are putatively conserved in the OPHs as well; an oxyanion hole and a catalytic triad were found at positions shifted downstream by about 50 amino acid residues from the corresponding positions in the PHB depolymerases. However, further work, including site-directed mutagenesis, is needed to determine the catalytic residues in OPHs.
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ACKNOWLEDGEMENTS |
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Received 24 September 2004;
revised 20 December 2004;
accepted 22 December 2004.
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