Production and Characterization of the Recombinant Sphingomonas chlorophenolica Pentachlorophenol 4-Monooxygenase

Pentachlorophenol 4-monooxygenase (PCP4MO) from Sphingomonas chlorophenolica is a flavoprotein that hydroxylates PCP in the presence of NADPH and oxygen. In order to investigate the structure and function of active site, recombinant PCP4MO (rePCP4MO) was produced in Escherichia coli as a glutathione...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2001-11, Vol.289 (1), p.161-166
Main Authors: Wang, Hong, Tiirola, Marja A., Puhakka, Jaakko A., Kulomaa, Markku S.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Binding Sites - genetics
DNA Primers - genetics
Endopeptidases - metabolism
Escherichia coli - genetics
flavin monooxygenase
GST fusion protein
Kinetics
Mixed Function Oxygenases - biosynthesis
Mixed Function Oxygenases - chemistry
Mixed Function Oxygenases - genetics
pentachlorophenol
pentachlorophenol 4-monooxygenase
Potyvirus - enzymology
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
Sphingomonas - enzymology
Sphingomonas - genetics
tetrachloro-p-hydroquinone
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Summary:Pentachlorophenol 4-monooxygenase (PCP4MO) from Sphingomonas chlorophenolica is a flavoprotein that hydroxylates PCP in the presence of NADPH and oxygen. In order to investigate the structure and function of active site, recombinant PCP4MO (rePCP4MO) was produced in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Moreover, a tobacco etch virus (TEV) protease cleavage site (EKLYFQG) was introduced into GST-PCP4MO and a his-tagged TEV protease was employed. Hence, a two-step purification protocol was developed which allowed obtaining 15–20 mg of rePCP4MO from 1 L culture. The rePCP4MO revealed identity with native enzyme by SDS–PAGE and N-terminal sequence analyses. Furthermore, a polyclonal PCP4MO antibody was produced with GST-PCP4MO and purified by immunoaffinity chromatography, where both the native and recombinant forms of PCP4MO showed interaction. However, rePCP4MO was identified as apoprotein with no evidence for a typical flavoprotein spectrum. The catalytic activity could be detected in the presence of FAD. The Km and Vmax values for PCP were 50 μM and 30 nmol/min/mg, respectively.
ISSN:0006-291X
1090-2104
DOI:10.1006/bbrc.2001.5915