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) comp onent of Rhodococcus globerulus P6 biphenyl dioxygenase. The a or beta subu nit of P6 oxygenase was exchanged with the corresponding subunit of Pseudom onas sp, strain LB400 or of Comamonas testosteroni B-356 to create new chim eras 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(B-356)beta(P6), and ht-alpha(B-356)beta(P6) ht-alpha(P6)beta(P6) ht-alpha(P6)beta(LB400) ht-al pha(P6)beta(B-356) were not expressed active in recombinant Escherichia col i cells carrying P6 bphA1 and bphA2, P6 bphA1 and LB400 bphE, or P6 bphA1 a nd B-356 bphE because the [2Fe-2S] Rieske cluster of P6 oxygenase a subunit was not assembled correctly in these clones. On the other hand ht-alpha(LB 400)beta(P6) and ht-alpha(B-356)beta(P6) were produced active in E, coli. F urthermore, active purified ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400) h t-alpha(P6)beta(B-356) showing typical spectra for Rieske-type proteins, we re obtained from Pseudomonas putida KT2440 carrying constructions derived f rom the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce h t-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 a! and a beta subunit recruited from distinct biphenyl dioxygenases is not determined specificall y by either one of the two subunits.