Abstract
2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBDO) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of the bphC gene from Comamonas sp. SMN4, which encodes 2,3-DBDO with His-tag, was cloned into a plasmid pQE30 in E. coli. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the purified active 2,3-DBDO showed a single band around 33 kDa, corresponding the molecular mass of 2,3-DBDO subunit. Two fractions around 170 and 100 kDa were separated in gel filtration chromatography, but only former one (the fraction of 170 kDa) has 2,3-DBDO activity. The 2,3-DBDO was reported as the polymeric protein consisted of eight subunits. However, the fraction corresponding octameric protein of 2,3-DBDO was not found in the gel filtration chromatography. The 2,3-DBDO was exhibited the maximum activity at pH 9.0 and was stable at pH 8.0, relatively. The circular dichroism (CD) data showed that 2,3-DBDO had an α-helical folding structures at neutral pHs ranged from pH 4.5 to pH 9.0. However, this high stable folding structure was converted to unfolded structure in acidic region (pH 2.5) or in high pH (pH 12.0). The enzyme was thermally stable and active up to 40 °C. The conformational data by CD spectra were consistent with the stability of 2,3-DBDO by checking the activity. The binding affinity (K m ) for 2,3-dihydroxybiphenyl, 3-metylcatechol, 4-methylcatechol and catechol was 11.7, 24 μM, 50 mM and 625 μM, respectively.
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References
Mondello FJ (1989) Cloning and expression in Escherichia coli of Pseudomonas strain LB400 genes encoding polychlorinated biphenyl degradation. J Bacteriol 171:1725–1732
Furukawa K, Suenaga H, Goto M (2004) Biphenyl dioxygenases: functional versatilities and directed evolution. J Bacteriol 186:5189–5196. doi:10.1128/JB.186.16.5189-5196.2004
Hayase N, Taira K, Furukawa K (1990) Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis, and expression in soil bacteria. J Bacteriol 172:1160–1164
Senda T, Kazuyuki S, Narita H, Yamamoto T, Kimbara K, Fukuda M, Sato M, Yano K, Mitsui Y (1996) Three-dimensional structures of free form and two substrate complexes of an extradiol ring-cleavage type dioxygenase, the BphC enzyme from Pseudomonas sp. strain KKS102. J Mol Biol 255:735–752. doi:10.1006/jmbi.1996.0060
Hein P, Powlowski J, Hurtubise Y, Ahmad D, Sylvestre M (1998) Biphenyl-associated meta-cleavage dioxygenases from Comamonas testosterone B-356. Can J Microbiol 44:42–49. doi:10.1139/w97-119
Sakai M (2002) Diversity of 2,3-dihydroxybiphenyl dioxygenase genes in a strong PCB degrader, Rhodocoddus sp. strain RAH1. J Biosci Bioeng 93:421–427
Furukawa K, Matsumura F (1976) Microbial metabolism of polychlorinated biphenyls. Studies on the relative degradability of polychlorinated biphenyl components by Alcaligenes sp. J Agric Food Chem 24:251–256. doi:10.1021/jf60204a002
On HY, Lee N, Kim YC, Kim CK, Kim YS, Park YK, Ka JO, Lee KS, Min KH (1998) Extradiol cleavage of two-ring structure of biphenyl and indole oxydation by biphenyl dioxygenase in Comamonas acidovorans. J Microbiol Biotechnol 8:264–269
Lee N, Lee JM, Min KH, Kwon DY (2003) The purification and characterization of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Comamonas sp. SMN4. J Microbiol Biotechnol 13:487–494
Eltis LD, Hofmann B, Hecht HJ, Lunsdorf H, Timmis KN (1993) Purification and crystallization of 2,3-dihydroxybiphenyl 1,2-dioxygenase. J Biol Chem 268:2727–2732
Khan AA, Nawaz MS, Cerniglia CE (1997) Rapid purification of an active recombinant His-tagged 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83. FEMS Microbiol Lett 62:1825–1830. doi:10.1111/j.1574-6968.1997.tb10404.x
Xiong F, Shuai JJ, Jin SF, Zhang J, Sun J, Peng RH, Yao QH (2012) Expression and characterization of a recombinant 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas. Mol Cell Toxicol 8:375–382. doi:10.1007/s13273-012-0046-0
Lee N, Kwon DY, Min KH (2003) Cloning and sequence analyses of a 2,3-dihydroxybiphenyl 1,2-dioxygenase gene (bphC) from Comamonas sp. SMN4 for phylogenetic and structural analysis. J Ind Microbiol Biotechnol 30:245–250. doi:10.1007/s10295-003-0039-z
Parath J, Carlson J, Olsson I, Belfrage G (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258:598–599. doi:10.1038/258598a0
Sambrook J, Russell DW (2001) Molecular cloning; a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. doi:10.1101/pdb.prot3901
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using Bicinchoninic acid. Anal Biochem 150:76–85. doi:10.1016/0003-2697(85)90442-7
Kwon DY, Kim PS (1994) The stabilizing effects of hydrophobic cores on peptide folding of bovine-pancreatic-trypsin-inhibitor folding-intermediate model. Eur J Biochem 223:631–636. doi:10.1111/j.1432-1033.1994.tb19035.x
Venyaminov SY, Yang JT (1996) Determination of protein secondary structure. In: Fasman GD (ed) Circular dichroism and the conformational analysis of biomolecules. Plenum Press, New York, pp 69–108. doi:10.1007/978-1-4757-2508-7-3
Furukawa K, Arimura N (1987) Purification and properties of 2,3-dihydroxybiphenyl dioxygenase from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes and Pseudomonas aeruginosa carrying the cloned bphC gene. J Bacteriol 169:924–927
Cantor CR, Schimmel PR (1980) Biophysical chemistry, part II: techniques for the study of biological structure and function. Freeman, San Francisco, pp 670–676. doi:10.1016/0307-4412(81)90143-6
Cao L, Cao Y, Wu G, Li M, Xu J, He J, Li S, Hong Q (2013) Cloning of three 2,3-dihydroxybiphenyl-1,2-dioxygenase genes from Achromobacter sp. BP3 and the analysis of their roles in the biodegradation of biphenyl. J Hazard Mater 261:246–252. doi:10.1016/j.jhazmat.2013.07.019
Chant A, Kraemer-Pecore CM, Watkin R, Kneale GG (2005) Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site. Protein Expr Purif 39:152–159. doi:10.1016/j.pep.2004.10.017
Kotake T, Matsuzawa J, Suzuki-Minakuchi C, Okada K, Nojiri H, Iwata K (2016) Purification and partial characterization of the extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase, in the fluorene degradation pathway from Rhodococcus sp. strain DFA3. Biosci Biotechnol Biochem 80:719–725. doi:10.1080/09168451.2015.1123605
Kim PS, Baldwin RL (1990) Intermediates in the folding reactions of small proteins. Ann Rev Biochem 59:631–660. doi:10.1146/annurev.bi.59.070190.003215
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This research was supported by the Korea Food Research Institute.
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Lee, N., Kwon, D.Y. Characteristics of a Recombinant 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from Comamonas sp. Expressed in Escherichia coli . Indian J Microbiol 56, 467–475 (2016). https://doi.org/10.1007/s12088-016-0599-z
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DOI: https://doi.org/10.1007/s12088-016-0599-z