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 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 K(m) and V(max) values for PCP were 50 microM and 30 nmol/min/mg, respectively.
Wang
H
H
Department of Biological and Environmental Science, University of Jyväskylä, FIN-40351 Jyväskylä, Finland.
Tiirola
M A
MA
Puhakka
J A
JA
Kulomaa
M S
MS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D017800:Potyvirus / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D006899:Mixed Function Oxygenases; C069781:pentachlorophenol monooxygenase; D010450:Endopeptidases; C099488:TEV protease