| dc.description.abstract | Biosynthesis of Indole Alkaloids in the Higher Plant Catharanthus roseus:
Is Cytochrome b System Involved in the Cytochrome P 450–Mediated Geraniol Hydroxylation ”
Indole alkaloids belong to a large family of structurally diverse compounds that include some of the most important plant derived medicinal agents discovered by humans. The chemistry of these natural products has been worked out in great detail. Although in vivo feeding experiments with labelled precursors have resulted in the elucidation of a generally accepted pathway for their biosynthesis, more details regarding the biosynthetic pathways of these complex organic molecules can perhaps be obtained by studying the individual steps involved in their biosynthesis at the cell free level.
The intermediacy of 8 hydroxy derivatives of geraniol and nerol in the biosynthesis of loganin-the non tryptamine moiety of indole alkaloids-was clearly demonstrated by earlier workers. These studies revealed that the 8 hydroxylation of geraniol and nerol is the first committed step in the biosynthesis of cyclopentanoid monoterpene glucosides, the non tryptamine moiety of indole alkaloids. It has been shown earlier that a membrane bound cytochrome P 450–dependent hydroxylase isolated from 5 day old C. roseus seedlings converts both geraniol and nerol to their respective 8 hydroxy derivatives in the presence of NADPH and O . However, NADH, when added along with NADPH, showed considerable synergistic effect.
The membrane bound hydroxylase has been solubilized, resolved into different components, and the hydroxylase activity was reconstituted using partially purified components. The reconstitution studies demonstrated the involvement of cytochrome P 450, NADPH–cytochrome c (P 450) reductase and phospholipid as components of the hydroxylase system. However, the cytochrome P 450 fraction used in the reconstitution studies contained significant levels of cytochrome b as well as NADH–cytochrome b reductase. The observed synergistic effect of NADH in the presence of NADPH and the presence of cytochrome b system in the cytochrome P 450 fraction led to speculation that the cytochrome b system may possibly participate in the cytochrome P 450–mediated geraniol hydroxylation reaction. In the mammalian system, there is evidence for the participation of cytochrome b in cytochrome P 450–mediated mixed function oxidase reactions.
In order to establish whether or not the cytochrome b system participates in the NADPH dependent cytochrome P 450–mediated geraniol hydroxylation reaction, an attempt was made to isolate and purify cytochrome P 450, cytochrome b and NAD(P)H–cytochrome c reductases. The results clearly indicated the components involved in the geraniol 8 hydroxylation reaction. The findings were further confirmed using antibodies raised against cytochrome b .
Microsomes were prepared from 5 day old C. roseus seedlings. CHAPS, a zwitterionic detergent, was used to solubilize cytochrome P 450 from the microsomes. The detergent solubilized microsomal fraction was subjected to hydrophobic and ion exchange chromatography. Following this procedure, two distinct forms of cytochrome P 450 were purified to significant levels. The highly purified forms (A I and B III) had specific contents of 2.4 and 3.5, respectively. Both forms had absorption peaks at 418, 522 and 556 nm in the oxidized form. Upon reduction with dithionite, the Soret band shifted to a longer wavelength. The reduced CO difference spectrum showed an absorption peak at 450 nm.
The microsomal NADH–ferricyanide (cytochrome b ) reductase was solubilized as described earlier. The reductase was purified by a combination of hydrophobic, ion exchange and affinity chromatography, yielding a specific activity of 51.4 nmol/min/mg. The purified reductase was homogeneous and estimated to have a molecular weight of 32,800. The oxidized form of the reductase showed a spectrum characteristic of a flavoprotein. The purified enzyme transferred electrons to other artificial electron acceptors such as DCPIP and PMS. Ferricyanide reduction was inhibited by sulfhydryl reagents such as NEM and p CMB.
Purified cytochrome b was reduced by NADH in the presence of the homogeneous NADH–ferricyanide reductase. The purified reductase also catalyzed electron transfer from NADH to cytochrome c in the presence of purified cytochrome b . These results clearly indicate that the purified enzyme was NADH–cytochrome b reductase of C. roseus.
Rabbits were immunized with purified cytochrome b . The anti cytochrome b serum inhibited the reduction of cytochrome c carried out with microsomes as well as with the reconstituted cytochrome b system [cytochrome b and NADH–cytochrome c (cytochrome b ) reductase]. The anti cytochrome b serum was used in the geraniol hydroxylase assays carried out with microsomes.
Reconstitution studies were carried out with purified components-cytochrome P 450, NADPH–cytochrome P 450 reductase, cytochrome b , NADH–cytochrome b reductase and phospholipid. Maximum geraniol hydroxylase activity was observed with cytochrome P 450, cytochrome P 450 reductase and phospholipid. The addition of cytochrome b and NADH–cytochrome b reductase did not enhance activity, clearly indicating the non participation of the cytochrome b system in the NADPH dependent cytochrome P 450–catalyzed geraniol hydroxylation reaction. Further, microsomal geraniol hydroxylase was not inhibited by anti cytochrome b serum.
Conclusion
The reconstitution studies carried out with highly purified components of the hydroxylase system, along with the results of immunochemical experiments, strongly suggest the non involvement of the cytochrome b system in the microsomal geraniol 8 hydroxylation reaction. | |
