Heterologous Expression and Characterization of the Purified Oxygenase Component of Rhodococcus globerulusP6 Biphenyl Dioxygenase and of Chimeras Derived from It

Abstract

In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase. The α or β 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-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, ht-αLB400βP6, and ht-αB-356βP6. ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356 were not expressed active in recombinant Escherichia coli cells carrying P6bphA1 and bphA2, P6 bphA1 and LB400bphE, or P6 bphA1 and B-356 bphEbecause the [2Fe-2S] Rieske cluster of P6 oxygenase α subunit was not assembled correctly in these clones. On the other hand ht-αLB400βP6 and ht-αB-356βP6 were produced active inE. coli. Furthermore, active purified ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, showing typical spectra for Rieske-type proteins, were obtained from Pseudomonas putidaKT2440 carrying constructions derived from the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce ht-proteins inPseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an α and a β subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.

Cell Surface Display of Organophosphorus Hydrolase in Pseudomonasputida Using an Ice‐Nucleation Protein Anchor

“…This bacterium is robust and ubiquitous in soils and is capable of using a variety of pollutants such as toluene and xylene. They have been shown to express heterogeneous genes and to colonize a variety of soils (13,14). P. putida KT2440 has also been classified as a nonpathogen by the National Institutes of Health.…”

Predominant Biphenyl Dioxygenase From Legacy Polychlorinated Biphenyl (PCB)-Contaminated Soil Is a Part of Unusual Gene Cluster and Transforms Flavone and Flavanone

“…To investigate the substrate preferences of the metagenome-extracted BPDO_AVS, the gene cluster bphAE_AVS was heterologously co-expressed with the gene cluster bphFGBC from Par. xenovorans LB400 ( Chebrou et al, 1999 ; Vézina et al, 2007 ). Such a whole-cell-based approach to infer the catalytic activity of various BPDOs has been already successfully applied ( Chebrou et al, 1999 ; Shindo et al, 2003 ; Barriault et al, 2004 ; Kagami et al, 2008 ; Pham et al, 2012 ; Toussaint et al, 2012 ).…”

“…xenovorans LB400 ( Chebrou et al, 1999 ; Vézina et al, 2007 ). Such a whole-cell-based approach to infer the catalytic activity of various BPDOs has been already successfully applied ( Chebrou et al, 1999 ; Shindo et al, 2003 ; Barriault et al, 2004 ; Kagami et al, 2008 ; Pham et al, 2012 ; Toussaint et al, 2012 ). The advantage of BPDO co-expression with the genes BphB and BphC is the possibility to easily verify the presence of the active BPDO enzyme pathway through the evolution of the yellow BP transformation product HOPDA ( Kumamaru et al, 1998 ; Barriault and Sylvestre, 2004 ).…”

“…The following plasmids were used: (i) for the expression of BPDO mined from the metagenome, pQE-31 plasmid was employed (ampicillin selection; Qiagen), (ii) for the complementation of BphF and BphG activities necessary for the BPDO function, pYH31-bphFGBC_LB400 plasmid was used, bearing the bphFGBC gene cluster from Par. xenovorans LB400 [kanamycin selection; original name pYH31[LB400- bphFGBC ], kindly provided by prof. Michel Sylvestre ( Chebrou et al, 1999 )].…”

Micro‐synteny conservation analysis revealed the evolutionary history of bacterial biphenyl degradation pathway

“…The BphA comprises four subunits (bphA1, bphA2, bphA3 and bphA4) and converts biphenyl into a dihydrodiol compound by adding two oxygen atoms into the biphenyl ring (Chain et al ., 2006). Meanwhile, these genes have been demonstrated to be crucial in the substrate specificity in biphenyl degradation (Hurtubise et al ., 1998; Chebrou et al ., 1999) and altering the affinity for their electron–acceptor proteins (Sevrioukova, 2005). The bphB encodes dihydrodiol dehydrogenase, which is involved in the upper pathway of biphenyl degradation (Furukawa et al ., 2004).…”