Heterologous expression and characterization of the purified oxygenase component of Rhodococcus globerulus P6 biphenyl dioxygenase and of chimeras derived from it
H. Chebrou et al., Heterologous expression and characterization of the purified oxygenase component of Rhodococcus globerulus P6 biphenyl dioxygenase and of chimeras derived from it, J BACT, 181(16), 1999, pp. 4805-4811
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.