Heterologous expression and characterization of the purified oxygenase component of Rhodococcus globerulus P6 biphenyl dioxygenase and of chimeras derived from it.
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.
Chebrou
H
H
INRS-Santé, Université du Québec, Pointe-Claire, Québec H9R 1G6, Canada.
Hurtubise
Y
Y
Barriault
D
D
Sylvestre
M
M
D001713:Biphenyl Compounds / Q000493:pharmacokinetics; D002874:Chromosome Mapping; D004926:Escherichia coli / Q000235:genetics; D005659:Fungicides, Industrial / Q000493:pharmacokinetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007506:Iron-Sulfur Proteins; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D011078:Polychlorinated Biphenyls / Q000493:pharmacokinetics; D016958:Pseudomonas putida / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D001713:Biphenyl Compounds; D005659:Fungicides, Industrial; D007506:Iron-Sulfur Proteins; D010105:Oxygenases; D011078:Polychlorinated Biphenyls; D011993:Recombinant Fusion Proteins; C012280:2,2'-dichlorobiphenyl; C009407:2,5,2',5'-tetrachlorobiphenyl; C010574:biphenyl; C029905:2,5-dichlorobiphenyl; C029906:3,3'-dichlorobiphenyl; C074161:biphenyl-2,3-dioxygenase