Novel Class of P450Redox Partner Fusion Enzymes

While the BM-3-type P450-CPR fusion system represented a novel class of P450 protein, the use of CPR enzymes to drive P450 catalysis was already well established. However, genome sequence data again alerted the community to a distinctive type of P450-redox partner fusion enzyme thus far found in Rhodococcus erythropolis, a number of species of the pathogen Burkholderia, and in R. metalli-durans. This enzyme has a P450 (N-terminal) fused to a C-terminal reductase domain resembling the well-characterized phthalate dioxygenase reductase (PDOR) enzyme that binds both FMN and 2Fe-2S cofactors and which transfers electrons to phthalate dioxygenase (PDO) [92] (Fig. 5.3b). The PDO from Burkholderia cepacia catalyzes the first step in the breakdown of the aromatic compound phthalate, forming a cis-dihydrodiol product [93]. Subsequent conversion by pathway enzymes leads to protocatechuate (3,4-dihydroxybenzoate), an intermediate in aromatic metabolic pathways [94]. The PDOR is reduced preferentially by NADH (over NADPH) and electrons are transferred through the FMN, then 2Fe-2S centers and onto the PDO [95].

The first such P450-PDOR fusion enzyme characterized originated from Rhodococcus sp. NCIMB 9784 [96]. The enzyme (CYP116B2) has been expressed in E. coli and purified, and its individual domains have also been isolated [96, 97]. While a bona fide physiological substrate has not been defined for this enzyme, it was shown to dealkylate 7-ethoxycoumarin at a low rate. This is an assay used widely for demonstrating P450 monooxygenase activity. The heme (P450) domain of this fusion P450 has >50 % amino acid identity with the class I P450 ThcB (CYP116A1) from Rhodococcus erythropolis. The gene encoding this class I P450 is adjacent to genes that likely encode its physiological redox partners - a fer-redoxin reductase (rhodocoxin reductase, thcD) and a 2Fe-2S ferredoxin (rhodo-coxin, thcC). CYP116A1 catalyzes the oxidative degradation of atrazine and thiocarbamate-based herbicides, including EPTC (S-ethyl dipropylthiocarbamate) [98]. CYP116B2 does not appear to have herbicide oxidation activity, but our recent studies of another member of the P450-PDOR fusion class, CYP116B1 from R.

metallidurans, indicate that this enzyme does catalyze such reactions (Warman, A.J. et al., unpublished data).

Research on this novel type of P450-redox partner fusion class is in its infancy, but genome analysis indicates that such enzymes are also present in various species of the pathogenic bacterium Burkholderia, and thus may be as similarly widespread in nature as are the CYP102 class of P450-CPR fusions. The biotech-nological potential of such enzymes will not be clear until more detailed analysis of their substrate selectivity, stability, and efficiency of their electron transport systems is undertaken. However, they may function in resistance to environmental toxins, and thus could be exploited in, for example, plant engineering for herbicide resistance.

Heal Yourself With Qi Gong

Heal Yourself With Qi Gong

Qigong also spelled Ch'i Kung is a potent system of healing and energy medicine from China. It's the art and science of utilizing breathing methods, gentle movement, and meditation to clean, fortify, and circulate the life energy qi.

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