Vol.
187,
September
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
16, 1992
609-614
MOLECULAR CLONING AND CHARACTERIZATION OF CATECHOL 2,3DIOXYGENASES FROM BIPHENYL/POLYCHLORINATED BIPHENYLSDEGRADING BACTERIA t
Hogil Chang, Jeongrai Lee, Seungkyun Roh, Seung Ryul Kim’, Kyung Rak Min, Chi-Kyung Kim2, Eung-Gook Kim’, and Youngsoo Kim* College of Pharmacy, ‘College of Medicine, and Xollege of Natural Sciences Chungbuk National University, KOREA Received
July
24,
1992
SUMMARY : Catechol 2,3-dioxygenases were cloned from &c&genes sp. KF711, Pseudomonm putida KF715, and Achromobacter xylosoxidans KF701 which are
biphenyl/polychlorinated biphenyls-degrading bacteria. All of the cloned enzymes were purified by preparative polyacrylamide gel electrophoresis (PAGE). The purified catechol 2,3-dioxygenases were significantly different from one another in ring-fission activities to catechol and its derivatives. The catechol 2,3-dioxygenase from Alcaligenes sp. KF711 exhibited higher ring-fission activity to 4-chlorocatechol than those from P. putidu KF715 and A. xylosoxidans KF701. In electrophoretic mobilities, the three enzymes were different from one another on nondenaturing PAGE but the same on SDS-PAGE. Q 1992RCademlc Press, Inc.
Biphenyl and polychlorinated biphenyls @iphenyl/PCB) are constituents of herbicides, lubricants, and antimicrobial agents. They are environmentally persistent and have potent toxicity to human being and animals (l-3). Several microorganisms have been identified to utilize biphenyl/PCB as carbon and energy sources for growth (4-9). Major catabolic pathway of the biphenyl/PCB is proceeded through dioxygenation and dehydrogenation in one of the two benzene rings to form 2,3-dihydroxybiphenyls, which are further catabolized through ring fission and hydrolysis to form benzoates (10-15). However, benzene-ring fission in the benzoates resulted from biphenyl/PCB catabolism is still unclear. In this work, we have cloned each catechol 2,3-dioxygenase (catechol:oxygen 2,3-oxidoreductase, EC 1.13.11.2) from biphenyl/PCB-degrading bacteria, namely Alcaligenes sp. KF711 and P. putida KF715. The cloned catechol2,3-dioxygenases are purified and compared with their enzymatic and physical properties. ’ This work was partially supported by a grant from Ministry of Education, KOREA. *To whom correspondence should be addressed at Department of Biochemistry, College of Pharmacy, Chungbuk National University, Cheongju 360-763 KOREA.
609
0006-291X/92 $4.00 Copyighr 0 1992 by Acudemic Press. Inc. All rights of reproduction in any form reserved.
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
MATERIALS
AND METHODS
RESEARCH
COMMUNICATIONS
Chemicals and Enzymes. General chemicals including catechol derivatives, ampicillin, and metal ions were obtained from Sigma Chemical Co. and Aldrich Chemical Co., Inc. Trypton, yeast extract, and agar were obtained from DIFCO. DNA modifying enzymes including restriction endonucleases and T4 DNA ligase were purchased from Boehringer Mannheim. Strains and Culture. Alcaligenes sp. KF711 and P. putida KF715 were supplied by K. Furukawa at Kyushu University, JAPAN. Escherichia culi HBlOl was used as a host for gene cloning. The Alcaligenes sp. KF711 and P. putida KF715 were grown in LB medium or MM0 salt containing 0.1% biphenyl as the sole carbon and energy source. E. coli HBlOl was grown in LB medium or LB medium supplimented with ampicillin (50 &ml). Bacterial
DNA Techniques. Chromosomal DNA was isolated by SDS-proteinase K lysis, and plasmid by alkali lysis (16). The chromosomal DNA and plasmid were further purified by CsCl-ethidium bromide ultracentrifugation (17). Restriction endonucleases and T4 DNA ligase were treated according to reaction conditions recommended by the enzyme suppliers. DNA was resolved on 0.7% agarose gel by electrophoresis with TAE buffer, and visualized by staining with ethidium bromide (17). Recombinant
Purification and Activity Assay of Catechol 2,3-Dioxygenase. E. coli HBlOl harboring each catechol 2,3-dioxygenase gene was grown to log phase in LB medium supplimented with ampicillin (50 &ml). Crude lysates were prepared from the each bacterial pellet by sonication followed by centrifugation. For purification of the cloned catechol 2,3-dioxygenases, crude lysates were subjected to acetone cut (35% to 55%), and then resolved on preparative 7.5%-polyacrylamide gel. Each catechol 2,3dioxygenase was eluted from the gel after specific staining with 0.5 M catechol. Catechol 2,3-dioxygenase activities were spectrophotometrically measured as described in elesewhere (18).
RESULTS
AND DISCUSSION
Biphenyl and polychlorinated biphenyls (biphenyl/PCB) released to the environment can cause considerable human health problems as a result of their toxicity and persistence. PCB with higher chlorination are more recalcitrant (19). However, several microorganisms capable of degrading biphenyl/PCB have been identified from the environment. Microbial degradation of biphenyl/PCB is initiated to form corresponding benzoates by sequential activities of biphenyl dioxygenase, dihydrodiol dehydrogenase, 2,3-dihydroxybiphenyl dioxygenase, and 2-hydroxy-6-oxo-6-phenyl hexa-2,4-dienoate hydrolase. However, benzene-ring fission in the resulting benzoates is not characterized well. We have cloned each catechol 2,3-dioxygenase encoded in chromosomal DNA of A. xylosoxidam KF701, Alcaligenes sp. KF711, and P. putida KF715. All of the bacterial strains can utilize biphenyl/PCB as carbon and energy sources for growth (7). Catechol 2,3-dioxygenase is involved in benzene-ring fission of catechol, a common 610
Vol.
187,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1234 pCNU401
I s
I s
I x
E
E
Ev KP 1
01
02
I ss C
St P I
St St
Kp I I
Pv
I
I c
P ss
Pv
Ev
I x
I
I
x
x
Hp
E
SP
A P I I
A I
1 Kb
pCNU501
Fig. 1. Gel pattern of recombinant plasmids pCNU501. Recombinant plasmids were resolved Lane 1 : DNA size marker. Lane 2 : pCNU201 pCNU401 digested with EcoRI. Lane 4 : pCNU501 DNA size marker is HindIII-digested lambda DNA 2.0 kb in size.
from pCMJ201, pCNU401, and agarose gel by electrophoresis. digested with BamHI. Lane 3 : digested with EcoRI and SphI. The with 23.1, 9.4, 6.6, 4.4, 2.3, and
on
Fig. 2. Physical maps of inserts in pCNU401 and pCNU501. Restriction endonucleases are AvaI (A), ClaI (C), EcoRI (E), EcoRV (Ev), HpaI (HP), KpnI (Kp), PstI (P), PvuII (I%), Sal1 (S), SphI (Sp), SstI (Ss), StuI (St), and XhoI (X).
intermediate in degradative pathways of aromatic compounds. The enzyme gene from A. xylusoxidans KF701 was cloned to a unique BamHI site of pBR322, and this clone was previously reported as pCNU201 (18,20). In this work, two catechol 2,3dioxygenase genesfrom Alcaligenes sp. KF711 and P. putida KF715 have been cloned to pBR322 and expressedinto E. coli HBlOl as follows. Chromosomal DNA from Alcaligenes sp. KF711 was digested with EcoRI, and chromosomal DNA from P. putidu KF715 with EcoRI and SphI. The digested chromosomalDNA was ligated to the same endonuclcasesite(s) of pBR322, and then transformed to E. coli HBlOl. These genomic libraries were independently screenedby ampicillin resistancefollowed by chromogenic selection with catechol spray. This chromogenic selection is yellow coloring by 2-hydroxymuconic semialdehydewhich is formed from colorless catechol by catechol 2,3-dioxygenase activity. One yellow clone was selectedfrom about 75,000 transformants in Alcaligenes sp. KF711 library, and this clone is designated as pCNU401. Another yellow clone was selectedfrom about 100,000 transformants in P. putidu KF715 library, and this clone is designatedaspCNU501. Recombinant plasmids from pCNU201, pCNU401, and pCNU501 were identified on agarosegel by electrophoresis,and are shown in Fig. 1. pCNU401 has a recombinant plasmid with 8.3-kb insert and 4.4-kb pBR322. pCNU501 has a recombinant plasmid with 4.6-kb insert and 3.8-kb pBR322. Physical maps of the pCNU401 and pCNU501 are shown in Fig. 2. The insert in pCNU401 is cut by ClaI, EcoRI, EcoRV, HpaI, PstI, SalI, SstI, and XhoI but not by AvaI, BamHI, BclI, HindIII, KpnI, and StuI. The insert in pCNU501 is cut by AvaI, EcoRI, EcoRV, KpnI, PstI, PvuII, SphI, and StuI but not by BamHI, CM, HindIII, HpaI, and XhoI. 611
Vol. 187, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 3. Activity staining of catechol 2,3-dioxygenases. Catechol 2,3-dioxygenases in crude lysates were resolved on nondenaturing polyacrylamide gel by electrophoresis, and then identified as yellow bands by specific staining with catechol. Lane 1 : pBR322. Lane 2 : pCNU201. Lane 3 : pCNU401. Lane 4 : pCNU501.
Catechol 2,3-dioxygenases from pCNU201, pCNU401, and pCNU501 were resolved on nondenaturing polyacrylamide gel by electrophoresis, and then identified as yellow bands by activity staining (Fig. 3). pCNU201, pCNU401, and pCNU501 do have catechol 2,3-dioxygenase but E. coli HBlOl containing pBR322 does not. Catechol 2,3-dioxygenase from pCNU401 exhibits different electrophoretic mobility from the others. Each cloned catechol 2,3-dioxygenase did exhibit the same electrophoretic mobility with its parental enzyme from A. xylosoxidans KF701, Alcaligenes sp. KF711, or P. putida KF715. The cloned catechol 2,3-dioxygenases were purified by acetone cut followed by preparative polyacrylamide gel electrophoresis. All of the purified catechol 2,3-dioxygenases exhibit single bands on SDS-polyacrylamide gel, and have the same electrophoretic mobility with 39,000 in size (Fig. 4). The purified catechol 2,3-dioxygenases exhibited differential ring-fission activities to catechol and its derivatives (Table 1). Catechol 2,3-dioxygenases from pCNU201 and pCNU501 exhibited the highest ring-fission activity to catechol, but the enzyme from pCNU401 to 4-methylcatechol among catechol, 4-chlorocatechol, 3-
BSA OA 4
c230 CA0 ST1
1
4'
h 1
3 5 7 9 11 Distance (cm)
Fig. 4. SDS-PAGE of catechol 2,3-dioxygenases purified from pCNU201, pCNU401, and pCNUSO1. The purified catechol 2,3-dioxygenases (C230) were resolved on SDS-polyacrylamide gel by electrophoresis, and then identified by staining with Coomassie brilliant blue R-250. Lane 1 : SDS-PAGE size marker. Lane 2 : pCNU201. Lane 3 : pCNU401. Lane 4 : pCNU501. The SDS-PAGE size marker is a mixture of bovine serum albumin (BSA), ovalbumin (OA), carbonic anhydrase (CA), and soybean trypsin inhibitor (STI) with 66.2, 45.0, 31.0, and 21.5 kd in size. Log M is a logarithm of molecular weight of each protein.
612
Vol.
187,
No.
Table
2, 1992
BIOCHEMICAL
1. Enzyme activity
AND
BIOPHYSICAL
of catechol 2,3-dioxygenases pCNU401, and pCNU501
RESEARCH
purified
from
COMMUNICATIONS
pCNU201,
Plasmids Substrates
Catechol 4-Chlorocatechol 3-Methylcatechol 4-Methylcatechol
pCNU201
pCNU401
pCNU501
100%(51.9)* 58% 46% 26%
100%(58.6)* 118% 88% 169%
100%(44.7)* 12% 10% 16%
Catechol2,3-dioxygenase activitiesto catecholderivativesareshownasrelativeactivity (%) to catecho as a substrate. One unit of the enzyme is defined as formation rate of l@oIe ring fission product per minute. Protein concentrationwas determinedby dye method. *Specificactivity asunit per mgof protein.
methylcatechol, and 4-methylcatechol. Catechol 2,3-dioxygenase from pCNU401 exhibited higher ring-fission activity to 4-chlorocatechol than those from pCNU201 and pCNU501. These results suggest that the catechol 2,3-dioxygenases may be isofunctional ring-fission enzymes in biphenyl/PCB-degrading bacteria.
REFERENCES 1. Smith, A.G. (1991) In Handbook of PesticideToxicology (W.J. Hayes, and E.R. Laws, Eds.) Vol. 2, pp. 731-916. Academic Press, Inc. 2. Kalmaz, E.V., and Kalmaz, G.D. (1979) Review Ecol. Model 6, 223-251. 3. Jones, G.R.N. (1989) Lancet 791-794. 4. Ahmed, M., and Focht, D.D. (1973) Can. J. Microbial. 19, 47-52. 5. Brown, J.F., Bedard, D.L., Brennan, M.J., Carnaham,J.C., Feng, H., and Wagner, R.E. (1987) Science236, 707-712. 6. Furukawa, K., and Chakraberty, A.M. (1982) App. Environ. Microbial. 44, 619626. 7. Furukawa, K., Hayase, N., Taira, K., and Tomizuka, N. (1989) J. Bacterial. 171, 5467-5472. 8. Nies, L., and Vogel, T.M. (1991) App. Environ. Microbial. 57, 2771-2774. 9. Barton, M.R., and Crawford, R.L. (1988) App. Environ. Microbial. 54, 594595. 10. Kahn, A.A., and Walia, S.K. (1991) App. Environ. Microbial. 57, 1325-1332. 11. Hayase, N., Taira, K., and Furukawa, K. (1990) J. Bacterial. 172, 1160-l 164. 12. Mondello, F.J. (1989) J. Bacterial. 171, 1725-1732. 13. Chaudhry, G.R., and Chapalamadugu,S. (1991) Microbiological Rev. 55, 59-79. 14. Kimbara, K., Hashimoto, T., Fukuda, M., Koana, T., Tagaki, M., Oishi, M., and Yano, K. (1989) J. Bacterial. 171, 2740-2747. 15. Ahmad, D., Masse, R., and Sylvestre, M. (1990) Gene 86, 53-61. 613
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
16. Birmboin, H.C., and Doly, J. (1979) Nucleic Acids Res. 7, 1513-1523. 17. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) In Molecular Cloning : A Laboratory Manual Vol 1-3, Cold Spring Harbor Laboratory Press, New York. 18. Kim, Y., Choi, B., Lee, J., Chang, H., and Min, K.R. (1992) Biochem. Biophys. Res. Comm. 183, 77-82. 19. Furukawa, K., Tomizuka, N., and Kamibayashi, A. (1979) App. Environ. Microbial. 38, 301-310. 20. Kim, Y., Choi, B., and Min, K.R. (1992) Arch. Pharm. Res. 15, 48-51.
614
187,
September
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
16, 1992
609-614
MOLECULAR CLONING AND CHARACTERIZATION OF CATECHOL 2,3DIOXYGENASES FROM BIPHENYL/POLYCHLORINATED BIPHENYLSDEGRADING BACTERIA t
Hogil Chang, Jeongrai Lee, Seungkyun Roh, Seung Ryul Kim’, Kyung Rak Min, Chi-Kyung Kim2, Eung-Gook Kim’, and Youngsoo Kim* College of Pharmacy, ‘College of Medicine, and Xollege of Natural Sciences Chungbuk National University, KOREA Received
July
24,
1992
SUMMARY : Catechol 2,3-dioxygenases were cloned from &c&genes sp. KF711, Pseudomonm putida KF715, and Achromobacter xylosoxidans KF701 which are
biphenyl/polychlorinated biphenyls-degrading bacteria. All of the cloned enzymes were purified by preparative polyacrylamide gel electrophoresis (PAGE). The purified catechol 2,3-dioxygenases were significantly different from one another in ring-fission activities to catechol and its derivatives. The catechol 2,3-dioxygenase from Alcaligenes sp. KF711 exhibited higher ring-fission activity to 4-chlorocatechol than those from P. putidu KF715 and A. xylosoxidans KF701. In electrophoretic mobilities, the three enzymes were different from one another on nondenaturing PAGE but the same on SDS-PAGE. Q 1992RCademlc Press, Inc.
Biphenyl and polychlorinated biphenyls @iphenyl/PCB) are constituents of herbicides, lubricants, and antimicrobial agents. They are environmentally persistent and have potent toxicity to human being and animals (l-3). Several microorganisms have been identified to utilize biphenyl/PCB as carbon and energy sources for growth (4-9). Major catabolic pathway of the biphenyl/PCB is proceeded through dioxygenation and dehydrogenation in one of the two benzene rings to form 2,3-dihydroxybiphenyls, which are further catabolized through ring fission and hydrolysis to form benzoates (10-15). However, benzene-ring fission in the benzoates resulted from biphenyl/PCB catabolism is still unclear. In this work, we have cloned each catechol 2,3-dioxygenase (catechol:oxygen 2,3-oxidoreductase, EC 1.13.11.2) from biphenyl/PCB-degrading bacteria, namely Alcaligenes sp. KF711 and P. putida KF715. The cloned catechol2,3-dioxygenases are purified and compared with their enzymatic and physical properties. ’ This work was partially supported by a grant from Ministry of Education, KOREA. *To whom correspondence should be addressed at Department of Biochemistry, College of Pharmacy, Chungbuk National University, Cheongju 360-763 KOREA.
609
0006-291X/92 $4.00 Copyighr 0 1992 by Acudemic Press. Inc. All rights of reproduction in any form reserved.
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
MATERIALS
AND METHODS
RESEARCH
COMMUNICATIONS
Chemicals and Enzymes. General chemicals including catechol derivatives, ampicillin, and metal ions were obtained from Sigma Chemical Co. and Aldrich Chemical Co., Inc. Trypton, yeast extract, and agar were obtained from DIFCO. DNA modifying enzymes including restriction endonucleases and T4 DNA ligase were purchased from Boehringer Mannheim. Strains and Culture. Alcaligenes sp. KF711 and P. putida KF715 were supplied by K. Furukawa at Kyushu University, JAPAN. Escherichia culi HBlOl was used as a host for gene cloning. The Alcaligenes sp. KF711 and P. putida KF715 were grown in LB medium or MM0 salt containing 0.1% biphenyl as the sole carbon and energy source. E. coli HBlOl was grown in LB medium or LB medium supplimented with ampicillin (50 &ml). Bacterial
DNA Techniques. Chromosomal DNA was isolated by SDS-proteinase K lysis, and plasmid by alkali lysis (16). The chromosomal DNA and plasmid were further purified by CsCl-ethidium bromide ultracentrifugation (17). Restriction endonucleases and T4 DNA ligase were treated according to reaction conditions recommended by the enzyme suppliers. DNA was resolved on 0.7% agarose gel by electrophoresis with TAE buffer, and visualized by staining with ethidium bromide (17). Recombinant
Purification and Activity Assay of Catechol 2,3-Dioxygenase. E. coli HBlOl harboring each catechol 2,3-dioxygenase gene was grown to log phase in LB medium supplimented with ampicillin (50 &ml). Crude lysates were prepared from the each bacterial pellet by sonication followed by centrifugation. For purification of the cloned catechol 2,3-dioxygenases, crude lysates were subjected to acetone cut (35% to 55%), and then resolved on preparative 7.5%-polyacrylamide gel. Each catechol 2,3dioxygenase was eluted from the gel after specific staining with 0.5 M catechol. Catechol 2,3-dioxygenase activities were spectrophotometrically measured as described in elesewhere (18).
RESULTS
AND DISCUSSION
Biphenyl and polychlorinated biphenyls (biphenyl/PCB) released to the environment can cause considerable human health problems as a result of their toxicity and persistence. PCB with higher chlorination are more recalcitrant (19). However, several microorganisms capable of degrading biphenyl/PCB have been identified from the environment. Microbial degradation of biphenyl/PCB is initiated to form corresponding benzoates by sequential activities of biphenyl dioxygenase, dihydrodiol dehydrogenase, 2,3-dihydroxybiphenyl dioxygenase, and 2-hydroxy-6-oxo-6-phenyl hexa-2,4-dienoate hydrolase. However, benzene-ring fission in the resulting benzoates is not characterized well. We have cloned each catechol 2,3-dioxygenase encoded in chromosomal DNA of A. xylosoxidam KF701, Alcaligenes sp. KF711, and P. putida KF715. All of the bacterial strains can utilize biphenyl/PCB as carbon and energy sources for growth (7). Catechol 2,3-dioxygenase is involved in benzene-ring fission of catechol, a common 610
Vol.
187,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1234 pCNU401
I s
I s
I x
E
E
Ev KP 1
01
02
I ss C
St P I
St St
Kp I I
Pv
I
I c
P ss
Pv
Ev
I x
I
I
x
x
Hp
E
SP
A P I I
A I
1 Kb
pCNU501
Fig. 1. Gel pattern of recombinant plasmids pCNU501. Recombinant plasmids were resolved Lane 1 : DNA size marker. Lane 2 : pCNU201 pCNU401 digested with EcoRI. Lane 4 : pCNU501 DNA size marker is HindIII-digested lambda DNA 2.0 kb in size.
from pCMJ201, pCNU401, and agarose gel by electrophoresis. digested with BamHI. Lane 3 : digested with EcoRI and SphI. The with 23.1, 9.4, 6.6, 4.4, 2.3, and
on
Fig. 2. Physical maps of inserts in pCNU401 and pCNU501. Restriction endonucleases are AvaI (A), ClaI (C), EcoRI (E), EcoRV (Ev), HpaI (HP), KpnI (Kp), PstI (P), PvuII (I%), Sal1 (S), SphI (Sp), SstI (Ss), StuI (St), and XhoI (X).
intermediate in degradative pathways of aromatic compounds. The enzyme gene from A. xylusoxidans KF701 was cloned to a unique BamHI site of pBR322, and this clone was previously reported as pCNU201 (18,20). In this work, two catechol 2,3dioxygenase genesfrom Alcaligenes sp. KF711 and P. putida KF715 have been cloned to pBR322 and expressedinto E. coli HBlOl as follows. Chromosomal DNA from Alcaligenes sp. KF711 was digested with EcoRI, and chromosomal DNA from P. putidu KF715 with EcoRI and SphI. The digested chromosomalDNA was ligated to the same endonuclcasesite(s) of pBR322, and then transformed to E. coli HBlOl. These genomic libraries were independently screenedby ampicillin resistancefollowed by chromogenic selection with catechol spray. This chromogenic selection is yellow coloring by 2-hydroxymuconic semialdehydewhich is formed from colorless catechol by catechol 2,3-dioxygenase activity. One yellow clone was selectedfrom about 75,000 transformants in Alcaligenes sp. KF711 library, and this clone is designated as pCNU401. Another yellow clone was selectedfrom about 100,000 transformants in P. putidu KF715 library, and this clone is designatedaspCNU501. Recombinant plasmids from pCNU201, pCNU401, and pCNU501 were identified on agarosegel by electrophoresis,and are shown in Fig. 1. pCNU401 has a recombinant plasmid with 8.3-kb insert and 4.4-kb pBR322. pCNU501 has a recombinant plasmid with 4.6-kb insert and 3.8-kb pBR322. Physical maps of the pCNU401 and pCNU501 are shown in Fig. 2. The insert in pCNU401 is cut by ClaI, EcoRI, EcoRV, HpaI, PstI, SalI, SstI, and XhoI but not by AvaI, BamHI, BclI, HindIII, KpnI, and StuI. The insert in pCNU501 is cut by AvaI, EcoRI, EcoRV, KpnI, PstI, PvuII, SphI, and StuI but not by BamHI, CM, HindIII, HpaI, and XhoI. 611
Vol. 187, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 3. Activity staining of catechol 2,3-dioxygenases. Catechol 2,3-dioxygenases in crude lysates were resolved on nondenaturing polyacrylamide gel by electrophoresis, and then identified as yellow bands by specific staining with catechol. Lane 1 : pBR322. Lane 2 : pCNU201. Lane 3 : pCNU401. Lane 4 : pCNU501.
Catechol 2,3-dioxygenases from pCNU201, pCNU401, and pCNU501 were resolved on nondenaturing polyacrylamide gel by electrophoresis, and then identified as yellow bands by activity staining (Fig. 3). pCNU201, pCNU401, and pCNU501 do have catechol 2,3-dioxygenase but E. coli HBlOl containing pBR322 does not. Catechol 2,3-dioxygenase from pCNU401 exhibits different electrophoretic mobility from the others. Each cloned catechol 2,3-dioxygenase did exhibit the same electrophoretic mobility with its parental enzyme from A. xylosoxidans KF701, Alcaligenes sp. KF711, or P. putida KF715. The cloned catechol 2,3-dioxygenases were purified by acetone cut followed by preparative polyacrylamide gel electrophoresis. All of the purified catechol 2,3-dioxygenases exhibit single bands on SDS-polyacrylamide gel, and have the same electrophoretic mobility with 39,000 in size (Fig. 4). The purified catechol 2,3-dioxygenases exhibited differential ring-fission activities to catechol and its derivatives (Table 1). Catechol 2,3-dioxygenases from pCNU201 and pCNU501 exhibited the highest ring-fission activity to catechol, but the enzyme from pCNU401 to 4-methylcatechol among catechol, 4-chlorocatechol, 3-
BSA OA 4
c230 CA0 ST1
1
4'
h 1
3 5 7 9 11 Distance (cm)
Fig. 4. SDS-PAGE of catechol 2,3-dioxygenases purified from pCNU201, pCNU401, and pCNUSO1. The purified catechol 2,3-dioxygenases (C230) were resolved on SDS-polyacrylamide gel by electrophoresis, and then identified by staining with Coomassie brilliant blue R-250. Lane 1 : SDS-PAGE size marker. Lane 2 : pCNU201. Lane 3 : pCNU401. Lane 4 : pCNU501. The SDS-PAGE size marker is a mixture of bovine serum albumin (BSA), ovalbumin (OA), carbonic anhydrase (CA), and soybean trypsin inhibitor (STI) with 66.2, 45.0, 31.0, and 21.5 kd in size. Log M is a logarithm of molecular weight of each protein.
612
Vol.
187,
No.
Table
2, 1992
BIOCHEMICAL
1. Enzyme activity
AND
BIOPHYSICAL
of catechol 2,3-dioxygenases pCNU401, and pCNU501
RESEARCH
purified
from
COMMUNICATIONS
pCNU201,
Plasmids Substrates
Catechol 4-Chlorocatechol 3-Methylcatechol 4-Methylcatechol
pCNU201
pCNU401
pCNU501
100%(51.9)* 58% 46% 26%
100%(58.6)* 118% 88% 169%
100%(44.7)* 12% 10% 16%
Catechol2,3-dioxygenase activitiesto catecholderivativesareshownasrelativeactivity (%) to catecho as a substrate. One unit of the enzyme is defined as formation rate of l@oIe ring fission product per minute. Protein concentrationwas determinedby dye method. *Specificactivity asunit per mgof protein.
methylcatechol, and 4-methylcatechol. Catechol 2,3-dioxygenase from pCNU401 exhibited higher ring-fission activity to 4-chlorocatechol than those from pCNU201 and pCNU501. These results suggest that the catechol 2,3-dioxygenases may be isofunctional ring-fission enzymes in biphenyl/PCB-degrading bacteria.
REFERENCES 1. Smith, A.G. (1991) In Handbook of PesticideToxicology (W.J. Hayes, and E.R. Laws, Eds.) Vol. 2, pp. 731-916. Academic Press, Inc. 2. Kalmaz, E.V., and Kalmaz, G.D. (1979) Review Ecol. Model 6, 223-251. 3. Jones, G.R.N. (1989) Lancet 791-794. 4. Ahmed, M., and Focht, D.D. (1973) Can. J. Microbial. 19, 47-52. 5. Brown, J.F., Bedard, D.L., Brennan, M.J., Carnaham,J.C., Feng, H., and Wagner, R.E. (1987) Science236, 707-712. 6. Furukawa, K., and Chakraberty, A.M. (1982) App. Environ. Microbial. 44, 619626. 7. Furukawa, K., Hayase, N., Taira, K., and Tomizuka, N. (1989) J. Bacterial. 171, 5467-5472. 8. Nies, L., and Vogel, T.M. (1991) App. Environ. Microbial. 57, 2771-2774. 9. Barton, M.R., and Crawford, R.L. (1988) App. Environ. Microbial. 54, 594595. 10. Kahn, A.A., and Walia, S.K. (1991) App. Environ. Microbial. 57, 1325-1332. 11. Hayase, N., Taira, K., and Furukawa, K. (1990) J. Bacterial. 172, 1160-l 164. 12. Mondello, F.J. (1989) J. Bacterial. 171, 1725-1732. 13. Chaudhry, G.R., and Chapalamadugu,S. (1991) Microbiological Rev. 55, 59-79. 14. Kimbara, K., Hashimoto, T., Fukuda, M., Koana, T., Tagaki, M., Oishi, M., and Yano, K. (1989) J. Bacterial. 171, 2740-2747. 15. Ahmad, D., Masse, R., and Sylvestre, M. (1990) Gene 86, 53-61. 613
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
16. Birmboin, H.C., and Doly, J. (1979) Nucleic Acids Res. 7, 1513-1523. 17. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) In Molecular Cloning : A Laboratory Manual Vol 1-3, Cold Spring Harbor Laboratory Press, New York. 18. Kim, Y., Choi, B., Lee, J., Chang, H., and Min, K.R. (1992) Biochem. Biophys. Res. Comm. 183, 77-82. 19. Furukawa, K., Tomizuka, N., and Kamibayashi, A. (1979) App. Environ. Microbial. 38, 301-310. 20. Kim, Y., Choi, B., and Min, K.R. (1992) Arch. Pharm. Res. 15, 48-51.
614
