Abstract
Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356. The enzyme comprises a two-subunit iron-sulfur protein (ISPBPH), a ferredoxin FERBPH, and a ferredoxin reductase REDBPH. REDBPH and FERBPH transfer electrons from NADH to an Fe-S active center of ISPBPH which activates molecular oxygen for insertion into the substrate. In this work B-356 ISPBPH complex and its alpha and beta subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system. His-tagged B-356 ISPBPH construction carrying a single His tail on the N-terminal portion of the alpha subunit was active. Its major features were compared to the untagged enzyme. In both cases, the native form is an alpha3beta3 heteromer, with each alphabeta unit containing a [2Fe-2S] Rieske center (epsilon455 = 8,300 M-1 cm-1) and a mononuclear Fe2+. Although purified His-tagged alpha subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation of this enzyme component and His-tagged beta subunit to reconstitute active ISPBPH was weak. However, when His-tagged alpha and beta subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISPBPH could be purified on Ni-nitrilotriacetic acid resin.
Highlights
Since active purified FERBPH and REDBPH were difficult to obtain from cell extracts of parental strains (1, 4), these enzyme components were purified from Escherichia coli recombinant clones using the His-bind QIAGEN system (1)
As shown below, purified Histagged ISPBPH was active in the Biphenyl dioxygenase (BPH dox) assay with added His-tagged FERBPH and His-tagged REDBPH
The Mr of the native ISPBPH and His-tagged ISPBPH were estimated by HPLC gel filtration and they were found to be of 234,000 and 186,000, respectively. These values indicate that the native conformation of B-356 ISPBPH is ␣33 and corroborate previously published data obtained for strain LB400-ISPBPH (4)
Summary
To obtain purified His-tagged B-356-ISPBPH, the coding region of B-356-bphAE was PCR-amplified from a cloned DNA fragment using the oligonucleotides I and IV. A 1.9kilobase DNA fragment (for bphAE), a 1.3-kilobase DNA fragment (for bphA), and a 0.6-kilobase DNA fragment (for bphE) containing the entire coding sequences were isolated and cloned into the compatible sites of pQE31 Constructions were such that the His tail added 13 amino acids (MRGSHHHHHHTDP) to the protein at its N-terminal portion. Mr values of native ISPBPH, His-tagged ISPBPH, and individual ␣ and  subunits were determined by HPLC using a Perkin-Elmer Series 3 chromatograph and a Waters Protein Pak 300 SW column (7.8 ϫ 300 mm). The activity was evaluated from measurement of substrate disappearance and metabolite production They were detected using a Perkin-Elmer LC95 UV/ visible detector set at 306 nm for 2,3-dihydro-2,3-dihydroxybiphenyl or 254 nm for biphenyl. The purification scheme for B2,3D will be described elsewhere
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
