The effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2

Abstract

The benzene dioxygenase from Pseudomonas putida MI] is a multicomponent complex comprising a flavoprotein reductase, a ferredoxin, and a terminal iron-sulfur protein (ISP). The catalytic activity of the isolated complex shows a nonlinear relationship with protein concentration in cell extracts, with the limiting factor for activity in vitro being ferredoxinBED. The relative levels of the three components were analyzed by using '251-labelled antibodies, and the functional molar ratio of ISPBED, ferredoxinBED, and reductaseBED was shown to be 1:0.9:0.8, respectively. The concentration of ferredoxinBED was confirmed by quantitative electron paramagnetic resonance spectroscopy of the 2Fe-2S centers in ferredoxinBED and 'SPBED of whole cells. These results demonstrate that the ferredoxinBED component is a limiting factor in dioxygenase activity in vitro. To determine if it is a limiting factor in vivo, a plasmid (pJRM606) overproducing ferredoxinBED was introduced into P. putida ML2. The benzene dioxygenase activity of this strain, measured in cell extracts, was fivefold greater than in the wild type, and the activity was linear with protein concentration in cell extracts above 2 mg/ml. Western blotting (immunoblotting) and electron paramagnetic resonance spectroscopic analysis confirmed an elevated level of ferredoxinBED protein' and active redox centers in the recombinant strain. However, in these cells, the increased level of ferredoxinBED had no effect on the overall rate of benzene oxidation by whole cells. Thus, we conclude that ferredoxinBED is not limiting at the high intracellular concentration (0.48 mM) found in cells.

Incorporation of carbon and nitrogen atoms into proteins measured by protein‐based stable isotope probing (Protein‐SIP)

“…The ability of P. putida ML2 to degrade benzene aerobically has been known for a long time 15. The organism oxidises benzene to catechol using benzene dioxygenase and cis‐benzene dihydrodiol dehydrogenase; both enzymes have been characterized in detail 16, 17. The knowledge of the protein sequences of these enzymes responsible for the initial steps of benzene degradation and the good growth behaviour with benzene as a substrate make the strain a perfect model organism for the Protein‐SIP study presented here.…”

“…To demonstrate the potential of Protein‐SIP, a model experiment was performed using Pseudomonas putida strain ML2, a strain which is well characterised for its ability to assimilate benzene 15–17. A similar experiment was conducted under anoxic conditions for anoxic growth with toluene as a substrate 18 .…”

Identification and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp. strain CA10

“…This might be due to weak expression as in the case of the bphA3 gene product in Pseudomonas pseudoalcaligenes KF707 (37). The component of benzene dioxygenase of P. putida ML2 has intracellular ␣ subunit of iron-sulfur protein/ferredoxin/reductase molar ratio of 1:0.45:0.8, suggesting that the multicomponent dioxygenase is an enzyme complex with a rather loose association between the oxygenase component, ferredoxin, and ferredoxin reductase (38). Although the expression level of CarAc in recombinant E. coli was low, the amount of CarAc expressed in E. coli might be sufficient to transport electrons to the terminal component, or CarAc might be more highly expressed in strain CA10 and able to transport electrons efficiently.…”

Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD+-dependent cis-benzene dihydrodiol dehydrogenase

“…Plasmid pRK2013, containing the transfer genes of RK2, was used to mobilize pHMT215-222 into P. putida F39/D. The recombinant P. putida F39/D strain that contained pHMT215-222 was selected on minimal medium agar containing 50 g of kanamycin per ml and benzene supplied in the vapor phase at 30ЊC as described previously (50).…”

“…The bedD gene together with the tac promoter was subcloned from pHMT222 into pJRD215 to form pHMT215-222, and the latter was introduced into P. putida 39/D by triparental mating. Transconjugants were selected for by streaking onto minimal salts agar plates supplemented with kanamycin and grown at 30ЊC in the presence of benzene or toluene vapor (50). The bedD gene product was found to complement the mutation in P. putida 39/D by allowing the latter to grow separately on benzene and on toluene as the sole carbon and energy source.…”