Cytochrome P450 oxygenases and their uses

Details Cytochrome P450 oxygenases and their uses Cytochrome P450 oxygenases and their uses

2002-05-08

2004-09-07

Achutamurthy, P. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S252300, C435S320100, C435S006120, C435S440000, C536S023200, C536S023100

Reexamination Certificate

active

06787343

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to oxygenase enzymes and methods of using such enzymes to produce Taxol (paclitaxel) and related taxoids.
INTRODUCTION
Cytochrome P450
Cytochrome P450 proteins are enzymes that have a unique sulfur atom ligated to the heme iron and that, when reduced, form carbon monoxide complexes. When complexed to carbon monoxide they display a major absorption peak (Soret band) near 450 nm. There are numerous members of the cytochrome P450 group including enzymes from both plants and animals. Members of the cytochrome P450 group can catalyse reactions such as unspecific monooxygenation, camphor 5-monooxygenation, steroid 1&bgr;-monooxygenation, and cholesterol monooxygenation (Smith et al. (eds.), Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, New York, 1997).
Paclitaxel
The complex diterpenoid Taxol (® Bristol-Myers Squibb; common name paclitaxel) (Wani et al.,
J. Am. Chem. Soc.
93:2325-2327, 1971) is a potent antimitotic agent with excellent activity against a wide range of cancers, including ovarian and breast cancer (Arbuck and Blaylock,
Taxol: Science and Applications
, CRC Press, Boca Raton, 397-415, 1995; Holmes et al.,
ACS Symposium Series
583:31-57, 1995). Taxol was isolated originally from the bark of the Pacific yew (
Taxus brevifolia
). For a number of years, Taxol was obtained exclusively from yew bark, but low yields of this compound from the natural source coupled to the destructive nature of the harvest, prompted new methods of Taxol production to be developed. Taxol currently is produced primarily by semisynthesis from advanced taxane metabolites (Holton et al.,
Taxol: Science and Applications
, CRC Press, Boca Raton, 97-121, 1995) that are present in the needles (a renewable resource) of various Taxus species. However, because of the increasing demand for this drug both for use earlier in the course of cancer intervention and for new therapeutic applications (Goldspiel,
Pharmacotherapy
17:110S-125S, 1997), availability and cost remain important issues. Total chemical synthesis of Taxol currently is not economically feasible. Hence, biological production of the drug and its immediate precursors will remain the method of choice for the foreseeable future. Such biological production may rely upon either intact Taxus plants, Taxus cell cultures (Ketchum et al.,
Biotechnol. Bioeng.
62:97-105, 1999), or, potentially, microbial systems (Stierle et al.,
J. Nat. Prod.
58:1315-1324, 1995). In all cases, improving the biological production yields of Taxol depends upon a detailed understanding of the biosynthetic pathway, the enzymes catalyzing the sequence of reactions, especially the rate-limiting steps, and the genes encoding these proteins. Isolation of genes encoding enzymes involved in the pathway is a particularly important goal, since overexpression of these genes in a producing organism can be expected to markedly improve yields of the drug.
The Taxol biosynthetic pathway is considered to involve more than 12 distinct steps (Floss and Mocek,
Taxol: Science and Applications
, CRC Press, Boca Raton, 191-208, 1995; and Croteau et al.,
Curr. Top. Plant Physiol.
15:94-104, 1996). However, very few of the enzymatic reactions and intermediates of this complex pathway have been defined. The first committed enzyme of the Taxol pathway is taxadiene synthase (Koepp et al.,
J. Biol. Chem.
270:8686-8690, 1995) that cyclizes the common precursor geranylgeranyl diphosphate (Hefner et al.,
Arch. Biochem. Biophys.
360:62-74, 1998) to taxadiene (FIG.
1
). The cyclized intermediate subsequently undergoes modification involving at least eight oxygenation steps, a formal dehydrogenation, an epoxide rearrangement to an oxetane, and several acylations (Floss and Mocek,
Taxol: Science and Applications
, CRC Press, Boca Raton, 191-208, 1995; and Croteau et al.,
Curr. Top. Plant Physiol.
15:94-104, 1996). Taxadiene synthase has been isolated from
T. brevifolia
and characterized (Hezari et al.,
Arch. Biochem. Biophys.
322:437-444, 1995), the mechanism of action defined (Lin et al.,
Biochemistry
35:2968-2977, 1996), and the corresponding cDNA clone isolated and expressed (Wildung and Croteau,
J. Biol. Chem.
271:9201-9204, 1996).
The second specific step of Taxol biosynthesis is an oxygenation (hydroxylation) reaction catalyzed by taxadiene-5&agr;(x-hydroxylase. The enzyme has been demonstrated in Taxus microsome preparations (Hefner et al.,
Methods Enzymol.
272:243-250, 1996), shown to catalyze the stereospecific hydroxylation of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5&agr;-ol (i.e., with double-bond rearrangement), and characterized as a cytochrome P450 oxygeniase (Hefner et al.,
Chemistry and Biology
3:479-489, 1996).
Since the first specific oxygenation step of the Taxol pathway was catalyzed by a cytochrome P450 oxygenase, it was logical to assume that subsequent oxygenation (hydroxylation and epoxidation) reactions of the pathway would be carried out by similar cytochrome P450 enzymes. Microsomal preparations (Hefner et al.,
Methods Enzymol.
272:243-250, 1996) were optimized for this purpose, and shown to catalyze the hydroxylation of taxadiene or taxadien-5&agr;-ol to the level of a pentaol (see
FIG. 2
for tentative biosynthetic sequence and structures based on the evaluation of taxane metabolite abundances (Croteau et al.,
Curr. Topics Plant Physiol.
15:94-104, 1995)), providing evidence for the involvement of at least five distinct cytochrome P450 taxane (taxoid) hydroxylases in this early part of the pathway (Hezari et al.,
Planta Med.
63:291-295, 1997).
Also, the remaining three oxygenation steps (C1 and C7 hydroxylations and an epoxidation at C4-C20; see
FIGS. 1 and 3
) likely are catalyzed by cytochrome P450 enzymes, but these reactions reside too far down the pathway to observe in microsomes by current experimental methods (Croteau et al.,
Curr. Topics Plant Physiol.
15:94-104, 1995; and Hezari et al.,
Planta Med.
63:291-295, 1997). Since Taxus (yew) plants and cells do not appear to accumulate taxoid metabolites bearing fewer than six oxygen atoms (i.e., hexaol or epoxypentaol) (Kingston et al.,
Prog. Chem. Org. Nat. Prod.
61:1-206, 1993), such intermediates must be rapidly transformed down the pathway, indicating that the oxygenations (hydroxylations) are relatively slow pathway steps and, thus, important targets for gene cloning.
Isolation of the genes encoding the oxygenases that catalyze the oxygenase steps of Taxol biosynthesis would represent an important advance in efforts to increase Taxol and taxoid yields by genetic engineering and in vitro synthesis.
SUMMARY OF THE INVENTION
The invention stems from the discovery of twenty-one amplicons (regions of DNA amplifier by a pair of primers using the polymerase chain reaction (PCR)). These amplicons can be used to identify oxygenases, for example, the oxygenases shown in SEQ ID NOS: 56-68 and 87-92 that are encoded by the nucleic acid sequences shown in SEQ ID NOS: 43-55 and 81-86. These sequences are isolated from the Taxus genus, and the respective oxygenases are useful for the synthetic production of Taxol and related taxoids, as well as intermediates within the Taxol biosynthetic pathway, and other taxoid derivatives. The sequences also can be used for the creation of transgenic organisms that either produce the oxygenases for subsequent in vitro use, or produce the oxygenases in vivo so as to alter the level of Taxol and taxoid production within the transgenic organism.
Another aspect of the invention provides the nucleic acid sequences shown in SEQ ID NOS: 1-21 and the corresponding amino acid sequences shown in SEQ ID NOS: 22-42, respectively, as well as fragments of these nucleic acid sequences and amino acid sequences. These sequences are useful for isolating the nucleic acid and amino acid sequences corresponding to full-length oxygenases. These amino acid sequences and nucleic acid sequences are also useful for creating specific binding agents that recognize the corresponding oxygenase

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