Department of Biotechnology, Tottori University, Tottori, Tottori 680-8552, Japan1
Marine Biotechnology Institute, Kamaishi Laboratory, Kamaishi, Iwate 026-0001, Japan2
Author for correspondence: Masayuki Shimao. Tel: +81 857 31 5268. Fax: +81 857 31 0881. e-mail: shimao{at}lemon.bio.tottori-u.ac.jp
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ABSTRACT |
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Keywords: oxidized polyvinyl alcohol hydrolase, PQQ-dependent polyvinyl alcohol dehydrogenase, pva operon, Pseudomonas sp. VM15C
Abbreviations: DIG, digoxigenin-3-O-succinyl--aminocaproyl-[5-(3-aminoallyl)-2'-deoxy-uridine-5'-triphosphate]; KPB, potassium phosphate buffer; OPH, oxidized polyvinyl alcohol hydrolase; PES, phenazine ethosulfate; PVA, polyvinyl alcohol; PVADH, polyvinyl alcohol dehydrogenase; PQQ, pyrroloquinoline quinone
The DDBJ accession number for the sequence reported in this paper is AB008494.
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INTRODUCTION |
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In this work, we cloned the gene (pvaB) encoding oxidized PVA hydrolase (OPH) involved in the second step of PVA degradation from the same strain, and determined the complete nucleotide sequence of the structural gene. OPH was expressed in Escherichia coli clones and characterized. OPH attacked oxidized PVA and cleaved the main chain of the macromolecule.
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METHODS |
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Bacterial strains and plasmids.
The sources of the bacterial strains and plasmids and their relevant characteristics are shown in Table 1.
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DNA manipulation.
The method for preparing total DNA from Pseudomonas sp. strain VM15C was as previously described (Shimao et al., 1996 ). Plasmid DNA isolation, transformation, restriction endonuclease digestion, ligation and agarose gel electrophoresis were performed as described by Sambrook et al. (1989)
.
Preparation of the probe for cloning the OPH-encoding gene.
A part (1200 bp) of the DNA sequence of the structural gene (Shimao et al., 1996 ) for PVADH was amplified with synthetic oligonucleotides, 5'-AAGATTCCGCCGATCGA-3' and 5'-GATGTTGTTCCAGCGAC-3', as primers by PCR. The DNA probe was prepared with digoxigenin-3-O-succinyl-
-aminocaproyl-[5-(3-aminoallyl)-2'-deoxy-uridine-5'-triphosphate] (DIG) via random-primed labelling by using a DIG labelling kit (Boehringer Mannheim) and the PCR product as the template.
Southern and colony hybridization.
Southern blotting was carried out with a semi-dry electroblotter (Owl Scientific). Southern and colony hybridization were accomplished with the DIG-labelled probe.
DNA sequencing.
The DNA sequences of both strands of pMSVA10 and its deletion derivatives were determined by using a dye terminator cycle sequencing kit and 373A DNA sequencer (Perkin-Elmer). Primers used were M13 universal primers and synthesized oligonucleotides specific to the sample DNA sequences.
Preparation and fractionation of the cell extract.
The cell extract was prepared by sonication and was fractionated by ultracentrifugation into membrane and soluble fractions as described previously (Shimao et al., 1996 ).
Enzyme assay.
PVADH activity was assayed as described previously (Shimao et al., 1996 ). A decrease in the degree of polymerization of PVA in a solution causes a decrease in the viscosity of the solution. Viscosity-decreasing activity was assayed in a reaction mixture containing 50 mM potassium phosphate buffer (KPB, pH 7·5), 150 mg PVA1500, 1 µM PQQ, 1 mM Ca2+, 0·1 mM phenazine ethosulfate (PES) and enzyme preparation, in a total volume of 15 ml. The reaction was carried out while reciprocally shaking (110 r.p.m.) at 30 °C. The viscosity of the reaction mixture was measured with an Ostwald viscometer at 30 °C, and the viscosity decrease (%) reflecting the PVA degradation was calculated by the following equation: Viscosity decrease (%)=100(
-
)/(
-
), where
and
are the values for the specific viscosity of the reaction mixture at reaction times 0 and t, respectively,
is that of the reaction mixture not containing the enzyme preparation, and
is that containing neither the enzyme preparation nor PVA.
Oxidized PVA was prepared by incubating a reaction mixture containing 50 mM KPB (pH 7·5), 150 mg PVA1500, 1 µM PQQ, 1 mM Ca2+ and 0·1 mM PES with cell extract of E. coli JM101(pMSVA9KN) containing 750 mU PVADH in a total volume of 15 ml at 30 °C while reciprocally shaking (110 r.p.m.) for 2 h. The reaction was stopped by inactivating PVADH activity with the addition of EDTA to the reaction mixture at a final concentration of 2 mM. A control solution containing no oxidized PVA (oxPVA solution) was similarly prepared except that PVA1500 was omitted from the reaction mixture.
OPH activity was assayed at 30 °C with a reaction mixture containing 100 µl 50 mM KPB (pH 7·5), 800 µl oxidized PVA solution and 100 µl enzyme preparation. The control reaction mixture contained oxPVA solution in place of oxidized PVA solution. At the start of the reaction and 30 min later, the A300 of a solution containing 100 µl 1 M sodium carbonate buffer (pH 10), 800 µl H2O and 100 µl reaction mixture was measured by using a solution containing 100 µl of the 1 M sodium carbonate buffer (pH 10), 800 µl H2O and 100 µl of the control reaction mixture as a reference. One unit of OPH activity is defined as the amount of enzyme required to decrease A300 by 1 unit min-1 in 1 ml of the reaction mixture.
The assay mixture for 4,6-nonanedione-hydrolysing activity contained 2 µl 4,6-nonanedione, 500 µl enzyme preparation and 500 µl 50 mM KPB (pH 7·5). The amount of 2-pentanone produced was determined by gas chromatography as described below. One unit of activity is defined as the amount of the enzyme needed to produce 1 µmol 2-pentanone min-1.
Analyses.
Protein concentration was determined by the method of Bradford (1976) with bovine serum albumin as the standard. SDS-PAGE was performed by the method of Laemmli (1970)
with a 12% gel. The amounts of 4,6-nonanedione, 2-pentanone and n-butyric acid were determined by gas chromatography. An aliquot (1 ml) of the reaction mixture was acidified with 100 µl 2 M HCl and extracted with 200 µl ethyl ether. After the ethyl ether layer had been separated by centrifugation at 16000 g for 1 min, 5 µl of the ethyl ether extract was injected into a gas chromatograph (GC-7A, Shimadzu) equipped with a flame-ionization detector and a glass column (3·2 mmx2·1 m) packed with Unisole F-200 30/60. Nitrogen was used as the carrier gas at a flow rate of 30 ml min-1, and the column temperature was set initially at 80 °C for 2 min, before being raised to 145 °C at a rate of 16 °C min-1. The injector and detector temperatures were maintained at 170 °C. Retention times were as follows under these conditions: 2-pentanone, 2·49 min; n-butyric acid, 9·45 min; and 4,6-nonanedione, 10·3 min.
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RESULTS |
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A Southern hybridization analysis of total DNA from Pseudomonas sp. strain VM15C with the DIG-labelled probe specific to the partial sequence of pvaA showed that SphI digestion generated a 5·7 kbp DNA fragment that hybridized to the probe. The restriction map for the DNA fragment previously cloned in pMSVA1KN (Fig. 1) and the expected hybridization site of the probe indicated that this 5·7 kbp SphI fragment contained the 1·9 kbp 5'-flanking region of the PVADH gene. In order to clone the SphI fragment, a subgenomic library was constructed in E. coli JM101 by ligating into the pUC18 vector the 47 kbp DNA fraction isolated from agarose gel after electrophoresis of SphI-digested total DNA. The library was subsequently screened by colony hybridization with the probe. Four positive colonies were obtained among the 300 examined. A plasmid designated as pMSVA10 was isolated from a positive clone and used for further study. A restriction map analysis showed that this plasmid had the 5·7 kbp SphI insert and that the cloned fragment had the entire structural gene for PVADH in the opposite orientation to the lac promoter (Fig. 1
). pMSVA11, which contained the SphI fragment in the opposite orientation to that in pMSVA10, was obtained via SphI digestion and religation. The PVADH activity was expressed in E. coli carrying each of these plasmids with or without IPTG induction (data not shown). This observation indicated the possibility that a native promoter for the PVADH gene was present on the SphI fragment.
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Some putative functional sites, the lipoprotein signal sequence and the lipase consensus sequence, were recognized in the deduced amino acid sequence of pvaB. Gly-Ile-Ser-Ser-Gly from positions 201 to 205 in the deduced amino acid sequence coincides with the lipase consensus sequence, Gly-X-Ser-X-Gly (Jendrossek et al., 1993 ). The amino-terminal sequence was a possible lipoprotein signal sequence. This sequence had positively charged Arg9 and Arg12 residues followed by a hydrophobic core from Val14 to Ala20, and Ala16-Leu-Ala-Leu-Ala-Ser-Val-Ala-Ser-Gly-Cys26, which represents the consensus sequence of a prokaryotic membrane lipoprotein lipid attachment site (Bairoch & Bucher, 1994
).
Function of the pvaB product
To investigate the function of the pvaB product, the viscosity-decreasing (PVA-degrading) activity was investigated by using cell extracts of E. coli JM101 carrying cloned genes (Fig. 2). The extract of cells carrying pMSVA12 (pvaA- pvaB+) did not show any viscosity-decreasing activity, and the activity of the extract of cells carrying pMSVA9KN (pvaA+ pvaB-) was also low. On the other hand, the activity expressed by pMSVA11 (pvaA+ pvaB+) was much greater than that of pMSVA9KN. The mixed cell extract of E. coli strains carrying pMSVA9KN and pMSVA12 also showed synergistic activity, being much greater than that of each cell extract and similar to that of the pMSVA11 cell extract. The activities of the cell extract from E. coli JM101(pMSVA11) and the mixed cell extract were also similar to that of the cell extract from Pseudomonas sp. strain VM15C. Furthermore, as described below, the JM101(pMSVA12) cell extract decreased the viscosity of PVA after treatment with PVADH. These findings indicated that the pvaB product is an enzyme which degrades oxidized PVA formed by PVADH.
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Degradation of oxidized PVA by OPH.
OPH hydrolysed 4,6-nonanedione, a ß-diketone compound. To confirm that OPH would degrade the putative ß-diketone structure formed in PVA by the PVADH reaction, a two-step procedure in which PVADH was active in the first step, while OPH attacked oxidized PVA in the second step, was carried out. PVADH requires Ca2+ for PQQ binding and can be completely inactivated by EDTA (Shimao et al., 1996 ), but OPH was not inhibited by EDTA, as described below. First, PVA was oxidized with the cell extract of E. coli JM101(pMSVA9KN) (pvaA+ pvaB-), then PVADH in the reaction mixture was inactivated with EDTA and, after this inactivation, the JM101(pMSVA12) (pvaA- pvaB+) cell extract was added to the reaction mixture and the whole was incubated (Fig. 4
). In the control reaction without the JM101(pMSVA12) cell extract, the rates of decrease of A300 measured at pH 10 and of viscosity were low. In the reaction with the JM101(pMSVA12) cell extract, however, both the parameters decreased rapidly, indicating that cleavage of the molecular chain of oxidized PVA was caused by the disruption of a ß-diketone group. The rate of decrease of A300 was proportional to the amount of the JM101(pMSVA12) cell extract added to the reaction mixture (data not shown). On the other hand, the oxidized PVA prepared as described above was degraded with cell extract of JM101(pMSVA12). The degraded PVA was isolated by the same procedure as described for the oxidized PVA. IR spectra of the degraded PVA showed a new but slight absorption at 1580 cm, which was not obderved for untreated PVA and the oxidized PVA, suggesting the occurrence of a small amount of carboxylate groups in the degraded PVA (data not shown).
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The activity of OPH for oxidized PVA and 4,6-nonanedione was compared. The JM101(pMSVA12) cell extract had OPH activity causing a decrease in A300 of 1·65 units min-1 (mg protein)-1. On the other hand, the 4,6-nonanedione-hydrolysing activity of the cell extract was 0·326 mU (mg protein)-1. Again, based on the assumption that the absorbance of the oxidized PVA and that of 4,6-nonanedione are similar, OPH can be estimated to be about 350 times more active toward oxidized PVA than 4,6-nonanedione.
Inhibitors
The effects of various enzyme inhibitors on the 4,6-nonanedione-hydrolysing activity and OPH activity in the cell extract of E. coli JM101(pMSVA12) were examined. The cell extract was treated with the inhibitors at 30 °C for 5 min, and the OPH and 4,6-nonanedione-hydrolysing activities were assayed. Each inhibitor was used at a final concentration of 1 mM. PMSF, a serine hydrolase inhibitor, completely inhibited both activities. This supports the hypothesis that the site having the lipase consensus sequence is the catalytic centre in OPH. On the other hand, semicarbazide hydrochloride, NaF, NaN3, KCN, the chelators 1,10-phenanthroline, iminodiacetic acid, 2,2'-bipyridine and EDTA, and the thiol reagents p-chloromercuribenzoic acid, iodoacetic acid and N-ethylmaleimide, did not significantly inhibit either activity.
Amino acid sequence similarity
The deduced amino sequence of OPH was compared with others in the SWISS-PROT database by using the FASTA program (Pearson & Lipman, 1988 ). The search did not indicate any protein showing high similarity to OPH, but showed that poly(hydroxyalkanoate) depolymerase (SWISS-PROT accession PHA_PSELE) of Pseudomonas lemoignei (Jendrossek et al., 1993
) had low homology to OPH (22% identity in a 205 amino acid overlap). Poly(hydroxyalkanoate) depolymerases have the lipase consensus sequence at their active sites. A BLAST search in a non-redundant sequence database at the National Center for Biotechnology Information, Bethesda, MD, USA, by using the BLASTP program (Altschul et al., 1990
), showed that the domain incorporating the lipase consensus sequence in OPH was similar to those of several poly(hydroxyalkanoate) depolymerases (GenBank accessions U68039, U68170, U10470) from Pseudomonas spp. (Briese et al., 1994
; Schirmer & Jendrossek, 1994
), esterase D (PIR locus S36781) (Ferreira et al., 1993
) from P. fluorescens, and acetylesterase (DDBJ accession D87681) from Aspergillus awamorii.
pvaB product and its subcellular localization
Cells, cell extract, and soluble and membrane fractions of E. coli JM101(pMSVA11) were analysed by SDS-PAGE (Fig. 5). The pvaB product, a 40 kDa polypeptide, was detected in the cells carrying pMSVA11, but not in the cells carrying pUC18. This molecular mass agrees with that calculated from the deduced amino acid sequence of the pvaB product. Although the product was seen in lanes of both the soluble and membrane fractions, the membrane fraction contained more of the product than the soluble fraction. Furthermore, the specific activity of OPH in the membrane fraction was several times higher than that in the soluble fraction (data not shown). These facts suggested that OPH may be a membrane-bound enzyme. SDS-PAGE could not detect the pvaA product as a distinguishable protein band. The pvaB product may have been expressed in a larger amount than the pvaA product.
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DISCUSSION |
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In accordance with the presence of the lipoprotein signal sequence, the pvaB product expressed in cells of E. coli JM101(pMSVA11) is suggested to be membrane-bound. The rather large amount of product detected in the soluble fraction was probably released from the membrane during sample preparation. The deduced amino acid sequence of the pvaA product, PVADH, also had a signal sequence and was a membrane-bound enzyme (Shimao et al., 1986 ). The presence of both enzymes at the bacterial cell surface may be necessary for the degradation of the macromolecule.
The lipase consensus sequence indicated that OPH may be a serine hydrolase and that Ser203 may be an active centre. This possibility was supported by the complete inhibition of activity by PMSF. Serine hydrolases generally have a charge-relay system, a catalytic triad consisting of serine, histidine and aspartate, and an oxyanion hole (Blow, 1976 ). In addition to the putative serine active centre, the charge-relay system may also be present in OPH, but the amino acids involved remain to be identified.
We have also found for the first time evidence indicating that the genes for PVADH and OPH, pvaA and B, constitute an operon, pvaBA. Its putative promoter was found upstream of pvaB, and its putative terminator downstream of pvaA.
The pvaB product, OPH, together with the pvaA product, PVADH, constitute an enzyme system for cleavage of the molecular chain of PVA. OPH degrades oxidized PVA formed by the action of PVADH, but does not degrade unoxidized PVA. As to the mechanism for PVA degradation, PVADH appears to introduce ß-diketone groups into the PVA molecule which are then attacked by OPH. Thus, cleavage of the PVA molecules proceeds through the formation of ß-diketone groups by the action of PVADH and then by hydrolysis catalysed by OPH (Fig. 6). PVA is a polyhydroxyl compound, but PVADH can also oxidize monohydroxyl secondary alcohols and give monoketone compounds (Shimao et al., 1986
). This raises the question of whether PVADH would form ß-diketones in PVA molecules fortuitously as a result of the random oxidation of hydroxyl groups, or preferentially as a result of the selective oxidation of vicinal pairs of hydroxyl groups. The linear increase in A300, and thus the linear increase in the ß-diketone structure during the oxidation of PVA by PVADH, indicates that PVADH carries out selective oxidation of vicinal pairs of hydroxyl groups in the PVA molecule. On the other hand, PVADH oxidized 4,6-nonanediol to 4,6-nonanedione via 4-hydroxy-6-oxononanone (data not shown). Thus PVADH oxidized the vicinal hydroxyl groups of the 4,6-nonanediol step by step. For the assumed selective oxidation of vicinal pairs of hydroxyl groups in PVA, an interesting mechanism may function but this remains to be studied in future work.
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OPH also hydrolysed the low-molecular-mass ß-diketone compound 4,6-nonanedione, producing n-butyric acid and 2-pentanone. Since OPH was not active toward 4-nonanone, OPH seems to be specific for ß-diketone compounds. A comparison of the ß-diketone-degrading activity of OPH towards oxidized PVA and low-molecular-mass ß-diketone compounds suggests that OPH is more specific for oxidized PVA than for the low-molecular-mass ß-diketone compounds.
In accordance with such specificity of OPH, no proteins similar to OPH have yet been reported. It was, however, found that OPH has slight similarity to some poly(hydroxyalkanoate) depolymerases that are also serine hydrolases having the lipase consensus sequence. Interestingly, both are enzymes that act on high-molecular-mass polymers. The slight but interesting similarity of OPH to poly(hydroxyalkanoate) depolymerases may have some bearing on this common property. However, the substrate specificity and dissimilarity of OPH to other types of protein may indicate that pvaB is evolutionarily unique.
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ACKNOWLEDGEMENTS |
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Received 22 June 1999;
revised 22 October 1999;
accepted 1 December 1999.