Kevin D. Walker

Taxol biosynthetic genes

The function and properties of heterologously expressed full-length cDNA clones, isolated from a Taxus cDNA library and specific to Taxol biosynthesis, are summarized. Recombinant enzymes are described that catalyze early steps of the pathway, including taxadiene synthase, taxadien-5alpha-ol-O-acetyltransferase and taxadien-5alpha-yl acetate 10beta-hydroxylase, and that catalyze late steps, including 10-deacetylbaccatin III-10beta-O-acetyltransferase and taxane 2alpha-O-benzoyltransferase. The properties of Taxus geranylgeranyl diphosphate synthase are also described; although this synthase does not mediate a committed step of Taxol biosynthesis, it does provide the universal plastidial diterpenoid precursor, geranylgeranyl diphosphate, for initiating Taxol biosynthesis.

The final acylation step in Taxol biosynthesis: Cloning of the taxoid C13-side-chain N-benzoyltransferase from Taxus

The formation of several acyl groups and an amide group of Taxol is catalyzed by regioselective CoA thioester-dependent acyltransferases. Several full-length acyltransferase sequences, obtained from a cDNA library constructed from mRNA isolated from Taxus cuspidata cells induced for Taxol production with methyl jasmonate, were individually expressed in Escherichia coli, from which a cDNA clone encoding a 3′-N-debenzoyl- 2′-deoxytaxol N-benzoyltransferase was identified. This recombinant enzyme catalyzes the stereoselective coupling of the surrogate substrate N-debenzoyl-(3′RS)-2′-deoxytaxol with benzoyl-CoA to form predominantly one 3′-epimer of 2′-deoxytaxol. The product 2′-deoxytaxol was confirmed by radio-HPLC,1H-NMR, and chemical ionization-MS. This enzymatic reaction constitutes the final acylation in the Taxol biosynthetic pathway. The full-length cDNA coding for the N-benzoyltransferase has an ORF of 1,323 nucleotides and encodes a 441-residue protein with a calculated molecular weight of 49,040. The recombinant enzyme expressed in E. coli has a pH optimum at 8.0, a kcat ≈ 1.5 ± 0.3 s−1 and Km values of 0.42 mM and 0.40 mM for the N-deacylated taxoid and benzoyl-CoA, respectively. In addition to improving the production yields of Taxol in genetically engineered host systems, this enzyme provides a means of attaching modified aroyl groups to taxoid precursors for the purpose of improving drug efficacy.

Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis

The structural pharmacophore of Taxol, responsible for binding the N terminus of the β-subunit of tubulin to arrest cell proliferation, comprises, in part, the 13-O-(N-benzoyl-3-phenylisoserinoyl) side chain. To identify the side chain transferase of Taxol biosynthesis, a set of transacylases obtained from an enriched cDNA library (constructed from mRNA isolated from Taxus cuspidata cells induced with methyl jasmonate for Taxol production) was screened. A cDNA clone (designated TAX7) encoding a taxoid C-13 O-phenylpropanoyltransferase was isolated which yielded a recombinant enzyme that catalyzes the selective 13-O-acylation of baccatin III with β-phenylalanoyl CoA as the acyl donor to form N-debenzoyl-2′-deoxytaxol. This enzymatic product was converted to 2′-deoxytaxol by chemical N-benzoylation, and the identity of this derivative was confirmed by spectrometric analyses. The full-length cDNA has an ORF of 1,335 bases and encodes a 445-aa protein with a calculated molecular weight of 50,546. Evaluation of kinetic parameters revealed Km values of 2.4 ± 0.5 μM and 4.9 ± 0.3 μM for baccatin III and β-phenylalanoyl-CoA, respectively. The pH optimum for the recombinant O-(3-amino-3-phenylpropanoyl)transferase is at 6.8. Identification of this clone completes acquisition of the five aroyl/acyltransferases involved in the biosynthesis of Taxol. Application of these transacylase genes in suitable host cells can improve the production yields of Taxol and could enable the preparation of second-generation Taxol analogs possessing greater bioactivity and improved water solubility.

Genetic transformation of mature Taxus: an approach to genetically control the in vitro production of the anticancer drug, taxol

Abstract This report demonstrates genetic transformation of two Taxus species. Taxus brevifolia and Taxus baccata, and expression of bacterial genes transferred into the plant genome by Agrobacterium tumefaciens. We used two strains of Agrobacterium tumefaciens (Bo542 and C58) to inoculate shoot segments of mature yew trees. The highest gall formation frequency (28.3%) was achieved with Taxus baccata using the Bo542 strain. Agrobacterium tumefaciens strain Bo542 induced significantly more galls (24%) than strin C58 (4%). Although we were able to induce on both Taxus species, Taxus baccata showed significantly higher susceptibility (14%) than Taxus brevifolia (7%). In contrast to untransformed callus cultures, the gall cell lines proliferated on phytormone-free medium and produced agropine as the result of T-DNA transfer. Southern blot analysis showed the presence of T-DNA sequence in the genome of these cell lines. Taxol and related taxane produced by the transgenic callus cultures were identified by mass spectrometry and immunoassay with monoclonal antibodies specific for taxol.

Genome sequencing and analysis of the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum NRRL 62431

BackgroundPaclitaxel (Taxol™) is an important anticancer drug with a unique mode of action. The biosynthesis of paclitaxel had been considered restricted to the Taxus species until it was discovered in Taxomyces andreanae, an endophytic fungus of T. brevifolia. Subsequently, paclitaxel was found in hazel (Corylus avellana L.) and in several other endophytic fungi. The distribution of paclitaxel in plants and endophytic fungi and the reported sequence homology of key genes in paclitaxel biosynthesis between plant and fungi species raises the question about whether the origin of this pathway in these two physically associated groups could have been facilitated by horizontal gene transfer.ResultsThe ability of the endophytic fungus of hazel Penicillium aurantiogriseum NRRL 62431 to independently synthesize paclitaxel was established by liquid chromatography-mass spectrometry and proton nuclear magnetic resonance. The genome of Penicillium aurantiogriseum NRRL 62431 was sequenced and gene candidates that may be involved in paclitaxel biosynthesis were identified by comparison with the 13 known paclitaxel biosynthetic genes in Taxus. We found that paclitaxel biosynthetic gene candidates in P. aurantiogriseum NRRL 62431 have evolved independently and that horizontal gene transfer between this endophytic fungus and its plant host is unlikely.ConclusionsOur findings shed new light on how paclitaxel-producing endophytic fungi synthesize paclitaxel, and will facilitate metabolic engineering for the industrial production of paclitaxel from fungi.

Stereochemistry and Mechanism of a Microbial Phenylalanine Aminomutase

The stereochemistry of a phenylalanine aminomutase (PAM) on the andrimid biosynthetic pathway in Pantoea agglomerans (Pa) is reported. PaPAM is a member of the 4-methylidene-1H-imidazol-5(4H)-one (MIO)-dependent family of catalysts and isomerizes (2S)-α-phenylalanine to (3S)-β-phenylalanine, which is the enantiomer of the product made by the mechanistically similar aminomutase TcPAM from Taxus plants. The NH(2) and pro-(3S) hydrogen groups at C(α) and C(β), respectively, of the substrate are removed and interchanged completely intramolecularly with inversion of configuration at the migration centers to form β-phenylalanine. This is a contrast to the retention of configuration mechanism followed by TcPAM.

Insights into the Mechanistic Pathway of the Pantoea agglomerans Phenylalanine Aminomutase

The biosynthesis of the hybrid peptide-polyketide antibiotic andrimid in Pantoea agglomerans requires the function of a phenylalanine aminomutase, AdmH (designated PaPAM), which converts (S)-a-phenylalanine to (S)-b-phenylalanine. PaPAM is a member of the class I lyase-like family that includes phenylalanine and tyrosine aminomutases (PAMs and TAMs , respectively), phenylalanine ammonia lyases (PALs), tyrosine ammonia lyases (TALs), and histidine ammonia lyases (HALs). PALs, TALs, and HALs produce aryl acrylates from the corresponding aminoacid substrate by the elimination of ammonia. The transformations that are performed by this family of enzymes are, in part, catalyzed by a 4-methylidene-1H-imidazol-5(4H)-one (MIO) prosthetic group. This cofactor is formed post-translationally from a tandem of active site residues, typically Ala-Ser-Gly, and is believed to function as an electrophile through its a/b-unsaturated keto functional group (Scheme 1). Two mechanisms for these transformations have been proposed; in the first, the amino group of the amino acid substrate acts as a nucleophile and attacks the methylidene of MIO through conjugate addition (earlier reports suggested that MIO was a dehydroalanyl moiety). Ammonia is subsequently expelled from the N-alkylated substrate through an a/b-elimination process, which results in the formation of an acrylate reaction intermediate that is released as such in the ammonia lyase reaction. Alternatively, the acrylate remains in the active site for amino group rebound to form the b-amino acid product in the aminomutase reaction. A second proposed mechanism suggests that p-electrons at the ortho-carbon atom of the phenyl ring of the substrate attack MIO, which acts as a Lewis acid, by Friedel–Crafts-like activation. The second process has been principally assigned to ammonia lyase reactions that yield unsaturated products by a/b-elimination, 13] but has also been implicated in the aminomutase reactions. The structures of several enzymes of the MIO-dependent family were characterized in earlier reports. The structure of Rhodobacter sphaeroides tyrosine ammonia lyase (RsTAL) in complex with the competitive inhibitor 2-aminoindan-2phosphonic acid, which is covalently bound by its amino group to MIO in the active site (Protein Data Bank (PDB) 2O7E) was determined. Yet, based on this structure, one of the MIO-based mechanisms was not suggested for the Scheme 1. Two proposed mechanisms for the conversion of substrate to product in a generic aminomutase: a) amino-group alkylation pathway and b) Friedel–Crafts aryl-alkylation pathway. An ammonia lyase reaction terminates at trans-cinnamate or trans-coumarate (X=H (phenylalanine) or OH (tyrosine), respectively).

Enhanced Conversion of Racemic α-Arylalanines to (R)-β-Arylalanines by Coupled Racemase/Aminomutase Catalysis

The Taxus phenylalanine aminomutase (PAM) enzyme converts several (S)-alpha-arylalanines to their corresponding (R)-beta-arylalanines. After incubating various racemic substrates with 100 microg of PAM for 20 h at 31 degrees C, each (S)-alpha-arylalanine was enantioselectively isomerized to its corresponding (R)-beta-product. With racemic starting materials, the ratio of (R)-beta-arylalanine product to the (S)-alpha-substrate ranged between 0.4 and 1.8, and the remaining nonproductive (R)-alpha-arylalanine became enriched. To utilize the (R)-alpha-isomer, the catalysis of a promiscuous alanine racemase from Pseudomonas putida (KT2440) was coupled with that of PAM to increase the production of enantiopure (R)-beta-arylalanines from racemic alpha-arylalanine substrates. The inclusion of a biocatalytic racemization along with the PAM-catalyzed reaction moderately increased the overall reaction yield of enantiopure beta-arylalanines between 4% and 19% (depending on the arylalanine), which corresponded to as much as a 63% increase compared to the turnover with the aminomutase reaction alone. The use of these biocatalysts, in tandem, could potentially find application in the production of chiral beta-arylalanine building blocks, particularly, as refinements to the process are made that increase reaction flux, such as by selectively removing the desired (R)-beta-arylalanine product from the reaction mixture.

Taxol Biosynthesis: Tyrocidine Synthetase A Catalyzes the Production of Phenylisoserinyl CoA and Other Amino Phenylpropanoyl Thioesters

In Taxus plants the biosynthesis of the pharmaceutical paclitaxel includes the transfer of β-amino phenylpropanoyls from coenzyme A to the diterpenoid baccatin III by an acyl CoA-dependent acyltransferase. Several enzymes on the pathway are known, yet a few remain unidentified, including the putative ligase that biosynthesizes key β-amino phenylpropanoyl CoAs. The multienzyme, nonribosomal peptide synthetase that produces tyrocidines contains a tridomain starter module tyrocidine synthetase A that normally activates (S)-α-Phe to an adenylate anhydride in the adenylation domain. The Phe moiety is then thioesterified by the pendent pantetheine of the adjacent thiolation domain. Herein, the adenylation domain was found to function as a CoA ligase, making α-, β-phenylalanyl, and phenylisoserinyl CoA. The latter two are substrates of a phenylpropanoyltransferase on the biosynthetic pathway of the antimitotic paclitaxel.

(S)-styryl-α-alanine used to probe the intermolecular mechanism of an intramolecular MIO-aminomutase

A Taxus canadensis phenylalanine aminomutase (TcPAM) catalyzes the isomerization of (S)-α- to (R)-β-phenylalanine, making (E)-cinnamate (~10%) as a byproduct at steady state. A currently accepted mechanism for TcPAM suggests that the amino group is transferred from the substrate to a prosthetic group comprised of an amino acid triad in the active site and then principally rebinds to the carbon skeleton of the cinnamate intermediate to complete the α-β isomerization. In contrast, when (S)-styryl-α-alanine is used as a substrate, TcPAM produces (2E,4E)-styrylacrylate as the major product (>99%) and (R)-styryl-β-alanine (<1%). Comparison of the rates of conversion of the natural substrate (S)-α-phenylalanine and (S)-styryl-α-alanine to their corresponding products (k(cat) values of 0.053 ± 0.001 and 0.082 ± 0.002 s(-1), respectively) catalyzed by TcPAM suggests that the amino group resides in the active site longer than styrylacrylate. To demonstrate this principle, inhibition constants (K(I)) for selected acrylates ranging from 0.6 to 106 μM were obtained, and each had a lower K(I) compared to that of (2E,4E)-styrylacrylate (337 ± 12 μM). Evaluation of the inhibition constants and the rates at which both the α/β-amino acids (between 7 and 80% yield) and styrylacrylate were made from a corresponding arylacrylate and styryl-α-alanine, respectively, by TcPAM catalysis revealed that the reaction progress was largely dependent on the K(I) of the acrylate. Bicyclic amino donor substrates also transferred their amino groups to an arylacrylate, demonstrating for the first time that ring-fused amino acids are productive substrates in the TcPAM-catalyzed reaction.