Heterologous expression and characterization of the purified oxygenase component of Rhodococcus globerulus P6 biphenyl dioxygenase and of chimeras derived from it

• Published in: Journal of bacteriology Vol. 181; no. 16; pp. 4805 - 4811
• Main Authors: Chebrou, H, Hurtubise, Y, Barriault, D, Sylvestre, M
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.08.1999
• Subjects: Bacteria; Bacteriology; Biochemistry; Biphenyl Compounds - pharmacokinetics; Chromosome Mapping; Enzymes; Enzymes and Proteins; Escherichia coli - genetics; Fungicides, Industrial - pharmacokinetics; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Iron-Sulfur Proteins; Oxygenases - genetics; Oxygenases - metabolism; Plasmids; Polychlorinated Biphenyls - pharmacokinetics; Pseudomonas putida - genetics; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Rhodococcus - enzymology; Rhodococcus - genetics; Rhodococcus globerulus; Substrate Specificity
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/JB.181.16.4805-4811.1999

In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase. The alpha or beta subunit of P6 oxygenase was exchanged with the corresponding subunit of Pseudomonas sp. strain LB400 or of Comamonas testosteroni B-356 to create new chimeras that were purified ht-proteins and designated ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356), ht-alpha(LB400)beta(P6), and ht-alpha(B-356)beta(P6). ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356) were not expressed active in recombinant Escherichia coli cells carrying P6 bphA1 and bphA2, P6 bphA1 and LB400 bphE, or P6 bphA1 and B-356 bphE because the [2Fe-2S] Rieske cluster of P6 oxygenase alpha subunit was not assembled correctly in these clones. On the other hand ht-alpha(LB400)beta(P6) and ht-alpha(B-356)beta(P6) were produced active in E. coli. Furthermore, active purified ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356), showing typical spectra for Rieske-type proteins, were obtained from Pseudomonas putida KT2440 carrying constructions derived from the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce ht-proteins in Pseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an alpha and a beta subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.