The Study On Rhodococcus Sp. R04Cytochrome P450Polymorphism And The Function Of CYP125

Cytochrome P450enzymes (P450s) are a superfamily of heme-containing proteins that introduce one oxygen atom derived from molecular oxygen into an organic molecule. These monooxygenases are widely distributed throughout eukaryotes and prokaryotes and play numerous physiological roles in the oxidative metabolism of endogenous and exogenous compounds.Cytochromes P450got their name from their character as hemoproteins and from their unusual spectral properties displaying a typical absorption maximum of the reduced CO-bound complex at450nm:cytochrome stands for a hemoprotein, P for pigment and450reflects the absorption peak of the CO complex at450nm. The ability of reduced P450to produce an absorption peak at450nm upon CO binding is still used for the estimation of the P450content.The genome of Rhodococcus sp. R04contains15different genes encoded cytochrome P450enzymes (P450s) at least. These P450s are thought to belong to13families classified by amino acid sequences and named by P450Nomenclature Committee Dr. David Nelson. The names of these enzymes are CYP102C3, CYP116B4, CYP123A6, CYP125A18, CYP125B2, CYP136C4, CYP136C5P, CYP150A16, CYP152F1, CYP153A32, CYP185B1, CYP186C1, CYP255A3, CYP255A4and CYP51B1. In fact, although the P450s sharing only14.64%identity of sequence in Rhodococcus sp. R04, all the P450enzymes have conserved helix K area "EXXR" and heme-binding area "FGXGXXXCXG". A simple phylogenetic analysis reveals that CYP125A18and CYP125B2are grouped with CYP125in Rhodococcus sp. RHA1and Mycobacterium tuberculosis, which can catalyze the terminal hydroxylation of C27-steroids, whereas, CYP125is similar with a-terpineol hydroxylase CYP108(P450terp).Genome annotation indicated that the genome of Rhodococcus sp. R04contain two different CYP125gene encoded CYP125A18and CYP125B2. CYP125A18and CYP125B2were cloned, expressed and characterised from Rhodococcus sp. R04. The UV-Vis spectrum of CYP125B2and CYP125A18exhibit the maximum absorption peaks at417.5and422nm, namely the Soret peak, respectively. CO-difference spectrum of CYP125B2and CYP125A18show the maximum absorption peaks of the Fe2+-CO complex at450and458nm, respectively, suggesting that they are the same properties with the other P450monooxygenase. Spectrophotometric titration of azole drug (Clotrimazole, Econazole Nitrate, Metronidazole, Ketoconazole,4-phenylimidazole and4-methyl-2-phenylimidazole) binding to CYP125B2and CYP125A18were carried out, and their Kd Value were determined. Analysis of azole drug binding to CYP indicated that the capability of azole drug binding to CYP125B2in sequence was4-phenylimidazole, Econazole Nitrate, Clotrimazole, Ketoconazole,4-methyl-2-phenylimid-azole, Metronidazole, however, the binding to CYP125A18in sequnce was Ketoconazole, Econazole Nitrate, Clotrim-azole,4-methyl-2-phenylimidazole, Metronidazole,4-phenylimidazole.We have found Rhodococcus sp. R04was able to grow in a minimal medium with cholesterol as sole carbon source and energy source. And the cholesterol could be degradated to92.3%in6days. It is reported that CYP125is essential for growth on cholesterol, steroids and aliphatic compounds. Accordingly, we speculate that the two different of CYP125A18and CYP125B2can play an important role in the course of cholesterol degradation in Rhodococcus sp. R04, but it is very necessary to make further efforts to investigate their functions.