Redirecting tropane alkaloid metabolism reveals pyrrolidine alkaloid diversity in Atropa belladonna.

Plant-specialized metabolism is complex, with frequent examples of highly branched biosynthetic pathways, and shared chemical intermediates. As such, many plant-specialized metabolic networks are poorly characterized. The N-methyl Δ1 -pyrrolinium cation is a simple pyrrolidine alkaloid and precursor of pharmacologically important tropane alkaloids. Silencing of pyrrolidine ketide synthase (AbPyKS) in the roots of Atropa belladonna (Deadly Nightshade) reduces tropane alkaloid abundance and causes high N-methyl Δ1 -pyrrolinium cation accumulation. The consequences of this metabolic shift on alkaloid metabolism are unknown. In this study, we utilized discovery metabolomics coupled with AbPyKS silencing to reveal major changes in the root alkaloid metabolome of A. belladonna. We discovered and annotated almost 40 pyrrolidine alkaloids that increase when AbPyKS activity is reduced. Suppression of phenyllactate biosynthesis, combined with metabolic engineering in planta, and chemical synthesis indicates several of these pyrrolidines share a core structure formed through the nonenzymatic Mannich-like decarboxylative condensation of the N-methyl Δ1 -pyrrolinium cation with 2-O-malonylphenyllactate. Decoration of this core scaffold through hydroxylation and glycosylation leads to mono- and dipyrrolidine alkaloid diversity. This study reveals the previously unknown complexity of the A. belladonna root metabolome and creates a foundation for future investigation into the biosynthesis, function, and potential utility of these novel alkaloids.

Biocatalysis of precursors to new-generation SB-T-taxanes effective against paclitaxel-resistant cancer cells.

A 10-O-deacetylbaccatin III 10-O-acetyltransferase biocatalyst from Taxus plants was expressed in bacteria whole-cells that were fed 10-O-deacetylbaccatin III and cyclopropane carboxylic acid. Product analysis by qualitative LC/ESI-MS suggested that the C10-acylated products baccatin III, 10-O-n-propionyl-10-O-deacetylbaccatin III, and 10-O-cyclopropanecarbonyl-10-O-deacetylbaccatin III were made in vivo. The results implied that the cells provided non-natural cyclopropanecarbonyl CoA, from a broad-specificity CoA ligase, and natural products, acetyl CoA and n-propionyl CoA, from reserves in the bacteria for use by acyltransferase to acylate 10-O-deacetylbaccatin III in vivo. The 10-acyl-10-O-deacetylbaccatin III are precursors used to synthesize new-generation paclitaxel analogs SB-T-1214 and SB-T-121303, which are effective against cancer cells resistant to paclitaxel and its drug derivatives. The kcat and KM of the acyltransferase for cyclopropanecarbonyl CoA (0.83 s-1, 0.15 M) and n-propionyl CoA (1.2 s-1, 0.15 M) guided scale-up efforts. The 10-acyl-10-O-deacetylbaccatin III analogs (∼45 mg each) were made in vitro by the acyltransferase when incubated with the commercial taxane 10-O-deacetylbaccatin III and synthesized cyclopropanecarbonyl or n-propionyl CoA. The structures of the 10-acyl products were verified by NMR analyses that confirmed C10 acylation of the taxane substrate. LC/ESI-MS/MS analysis also supported the identities of the biocatalyzed products. This effort provides a biocatalysis framework to produce new-generation taxane precursors.

CoA recycling by a benzoate coenzyme A ligase in cascade reactions with aroyltransferases to biocatalyze paclitaxel analogs.

A Pseudomonas CoA ligase (BadA) biocatalyzed aroyl CoA thioesters used by a downstream N-benzoyltransferase (NDTNBT) in a cascade reaction made aroyl analogs of the anticancer drug paclitaxel. BadA kept the high-cost aroyl CoA substrates at saturation for the downstream NDTNBT by recycling CoA when it was added as the limiting reactant. A deacylated taxane substrate N-debenzoyl-2'-deoxypaclitaxel was converted to its benzoylated product at a higher yield, compared to the converted yield in assays in which the BadA ligase chemistry was omitted, and benzoyl CoA was added as a cosubstrate. The resulting benzoylated product 2'-deoxypaclitaxel was made at 196% over the theoretical yield of product that could be made from the CoA added at 50 µM, and the cosubstrates benzoic acid (100 µM), and N-debenzoyl-2'-deoxypaclitaxel (500 µM) added in excess. In addition, a 2-O-benzoyltransferase (mTBT) was incubated with BadA, aroyl acids, CoA, a 2-O-debenzoylated taxane substrate, and cofactors under the CoA-recycling conditions established for the NDTNBT/BadA cascade. The mTBT/BadA combination also made various 2-O-aroylated products that could potentially function as next-generation baccatin III compounds. These ligase/benzoyltransferase cascade reactions show the feasibility of recycling aroyl CoA thioesters in vitro to make bioactive acyl analogs of paclitaxel precursors.

Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities.

Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over ß-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the ß-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

Paclitaxel Biosynthesis: Adenylation and Thiolation Domains of an NRPS TycA PheAT Module Produce Various Arylisoserine CoA Thioesters.

Structure-activity relationship studies show that the phenylisoserinyl moiety of paclitaxel (Taxol) is largely necessary for the effective anticancer activity. Several paclitaxel analogues with a variant isoserinyl side chain have improved pharmaceutical properties versus those of the parent drug. To produce the isoserinyl CoAs as intermediates needed for enzyme catalysis on a semibiosynthetic pathway to paclitaxel analogues, we repurposed the adenylation and thiolation domains (Phe-AT) of a nonribosomal peptide synthetase (TycA) so that they would function as a CoA ligase. Twenty-eight isoserine analogue racemates were synthesized by an established procedure based on the Staudinger [2+2] cycloaddition reaction. Phe-AT converted 16 substituted phenylisoserines, one ß-(heteroaryl)isoserine, and one ß-(cyclohexyl)isoserine to their corresponding isoserinyl CoAs. We imagine that these CoA thioesters can likely serve as linchpin biosynthetic acyl donors transferred by a 13-O-acyltransferase to a paclitaxel precursor baccatin III to make drug analogues with better efficacy. It was also interesting to find that an active site mutant [Phe-AT (W227S)] turned over 2-pyridylisoserine and the sterically demanding p-methoxyphenylisoserine substrates to their CoA thioesters, while Phe-AT did not. This mutant is promising for further development to make 3-fluoro-2-pyridylisoserinyl CoA, a biosynthetic precursor of the oral pharmaceutical tesetaxel used for gastric cancers.

Biocatalysis of a Paclitaxel Analogue: Conversion of Baccatin III to N-Debenzoyl-N-(2-furoyl)paclitaxel and Characterization of an Amino Phenylpropanoyl CoA Transferase.

In this study, we demonstrate an enzyme cascade reaction using a benzoate CoA ligase (BadA), a modified nonribosomal peptide synthase (PheAT), a phenylpropanoyltransferase (BAPT), and a benzoyltransferase (NDTNBT) to produce an anticancer paclitaxel analogue and its precursor from the commercially available biosynthetic intermediate baccatin III. BAPT and NDTNBT are acyltransferases on the biosynthetic pathway to the antineoplastic drug paclitaxel in Taxus plants. For this study, we addressed the recalcitrant expression of BAPT by expressing it as a soluble maltose binding protein fusion (MBP-BAPT). Further, the preparative-scale in vitro biocatalysis of phenylisoserinyl CoA using PheAT enabled thorough kinetic analysis of MBP-BAPT, for the first time, with the cosubstrate baccatin III. The turnover rate of MBP-BAPT was calculated for the product N-debenzoylpaclitaxel, a key intermediate to various bioactive paclitaxel analogues. MBP-BAPT also converted, albeit more slowly, 10-deacetylbaccatin III to N-deacyldocetaxel, a precursor of the pharmaceutical docetaxel. With PheAT available to make phenylisoserinyl CoA and kinetic characterization of MBP-BAPT, we used Michaelis-Menten parameters of the four enzymes to adjust catalyst and substrate loads in a 200-µL one-pot reaction. This multienzyme network produced a paclitaxel analogue N-debenzoyl-N-(2-furoyl)paclitaxel (230 ng) that is more cytotoxic than paclitaxel against certain macrophage cell types. Also in this pilot reaction, the versatile N-debenzoylpaclitaxel intermediate was made at an amount 20-fold greater than the N-(2-furoyl) product. This reaction network has great potential for optimization to scale-up production and is attractive in its regioselective O- and N-acylation steps that remove protecting group manipulations used in paclitaxel analogue synthesis.

Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana.

Despite the importance of riboflavin as the direct precursor of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the physiologically relevant catalyst dephosphorylating the riboflavin biosynthesis pathway intermediate 5-amino-6-ribitylamino-2,4(1H,3H) pyrimidinedione 5'-phosphate (ARPP) has not been characterized from any organism. By using as the query sequence a previously identified plastidial FMN hydrolase AtcpFHy1 (At1g79790), belonging to the haloacid dehalogenase (HAD) superfamily, seven candidates for the missing ARPP phosphatase were found, cloned, recombinantly expressed, and purified. Activity screening showed that the enzymes encoded by AtcpFHy1, At4g11570, and At4g25840 catalyze dephosphorylation of ARPP. AtcpFHy1 was renamed AtcpFHy/PyrP1, At4g11570 and At4g25840 were named AtPyrP2 and AtGpp1/PyrP3, respectively. Subcellular localization in planta indicated that AtPyrP2 was localized in plastids and AtGpp1/PyrP3 in mitochondria. Biochemical characterization of AtcpFHy/PyrP1 and AtPyrP2 showed that they have similar Km values for the substrate ARPP, with AtcpFHy/PyrP1 having higher catalytic efficiency. Screening of 21 phosphorylated substrates showed that AtPyrP2 is specific for ARPP. Molecular weights of AtcpFHy/PyrP1 and AtPyrP2 were estimated at 46 and 72 kDa, suggesting dimers. pH and temperature optima for AtcpFHy/PyrP1 and AtPyrP2 were ~7.0-8.5 and 40-50°C. T-DNA knockout of AtcpFHy/PyrP1 did not affect the flavin profile of the transgenic plants, whereas silencing of AtPyrP2 decreased accumulation of riboflavin, FMN, and FAD. Our results strongly support AtPyrP2 as the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana. The identification of this enzyme closes a long-standing gap in understanding of the riboflavin biosynthesis in plants.

A Tyrosine Aminomutase from Rice (Oryza sativa) Isomerizes (S)-α- to (R)-ß-Tyrosine with Unique High Enantioselectivity and Retention of Configuration.

A recently discovered 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent tyrosine aminomutase (OsTAM) from rice [Yan, J., et al. (2015) Plant Cell 27, 1265] converts (S)-α-tyrosine to a mixture of (R)- and (S)-ß-tyrosines, with high (94%) enantiomeric excess, which does not change with pH, like it does for two bacterial TAMs. The K(M) of 490 µM and the k(cat) of 0.005 s(-1) are similar for other TAM enzymes. OsTAM is unique and also catalyzes (R)-ß- from (S)-α-phenylalanine. OsTAM principally retains the configuration at the reactive C(α) and C(ß) centers during catalysis much like the phenylalanine aminomutase on the Taxol biosynthetic pathway in Taxus plants.

Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity.

A benzoate CoA ligase (BadA), isolated from the bacterium Rhodopseudomonas palustris, catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids. Herein, apparent Michaelis constants K(app)cat and K(app)M were determined for an expanded array of 31 substrates chosen to systematically probe the active site architecture of the enzyme and provide a baseline for expansion of wild-type substrate specificity. Acyl CoA products were observed for 25 of the 31 substrates; in general, BadA converted ortho-substituted substrates better than the corresponding meta and para regioisomers, and the turnover number was more affected by steric rather than electronic effects. The kinetic data are interpreted in relation to six crystal structures of BadA in complex with several substrates and a benzoyl-AMP reaction intermediate. In contrast to other known natural substrate-bound benzoate ligase structures, all substrate-bound BadA structures adopted the thiolation conformation instead of the adenylation conformation. We also observed all the aryl carboxylates to be uniquely oriented within the active site, relative to other structures. Together, the kinetics and structural data suggested a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation. On the basis of this hypothesis and the structural data, sterically demanding active site residues were mutated, and the substrate specificity was expanded substantially versus that of BadA. Novel activities were seen for substrates with larger substituents, including phenyl acetate. Additionally, the mutant Lys427Ala identified this nonconserved residue as essential for the thiolation step of BadA, but not adenylation. These variously acylated CoAs can serve as novel substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogues of bioactive natural products.

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

BACKGROUND: Paclitaxel (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. RESULTS: The 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. CONCLUSIONS: Our 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.

A bacterial tyrosine aminomutase proceeds through retention or inversion of stereochemistry to catalyze its isomerization reaction.

ß-Amino acids are biologically active compounds of interest in medicinal chemistry. A class I lyase-like family of aminomutases isomerizes (S)-α-arylalanines to the corresponding ß-amino acids by exchange of the NH2/H pair. This family uses a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) group within the active site to initiate the reaction. The absolute stereochemistry of the product is known for an MIO-dependent tyrosine aminomutase from Chondromyces crocatus (CcTAM) that isomerizes (S)-α-tyrosine to (R)-ß-tyrosine. To evaluate the cryptic stereochemistry of the CcTAM mechanism, (2S,3S)-[2,3-(2)H2]- and (2S,3R)-[3-(2)H]-α-tyrosine were stereoselectively synthesized from unlabeled (or [(2)H]-labeled) (4'-hydroxyphenyl)acrylic acids by reduction with D2 (or H2) gas and a chiral Rh-Prophos catalyst. GC/EIMS analysis of the [(2)H]-ß-tyrosine biosynthesized by CcTAM revealed that the α-amino group was transferred to Cß of the phenylpropanoid skeleton with retention of configuration. These labeled substrates also showed that the pro-(3S) proton exchanges with protons from the bulk media during its migration to Cα during catalysis. (1)H- and (2)H NMR analyses of the [(2)H]-ß-tyrosine derived from (2S)-[3,3-(2)H2]-α-tyrosine by CcTAM catalysis showed that the migratory proton attached to Cα of the product also with retention of configuration. CcTAM is stereoselective for (R)-ß-tyrosine (85%) yet also forms the (S)-ß-tyrosine enantiomer (15%) through inversion of configuration at both migration termini, as described herein. The proportion of the (S)-ß-isomer made by CcTAM during steady state interestingly increased with solvent pH, and this effect on the proposed reaction mechanism is also discussed.

Assessing the deamination rate of a covalent aminomutase adduct by burst phase analysis.

Burst-phase kinetic analysis was used to evaluate the deamination rate of the aminated-methylidene imidazolone (NH(2)-MIO) adduct of a Taxus phenylalanine aminomutase. The kinetic parameters were interrogated by a non-natural substrate (S)-styryl-α-alanine that yielded a chromophoric styrylacrylate product upon deamination by the aminomutase. Transient inactivation of the enzyme by the NH(2)-MIO adduct intermediate resulted in an initial burst of product, with reactivation by deamination of the adduct. This study validated the rate constants of a kinetic model demonstrating that the NH(2)-MIO adduct and cinnamate intermediate are sufficiently retained to catalyze the natural α- to ß-phenylalanine isomerization.

(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)-ß-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-ß 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-ß-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/ß-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.

Mechanistic, mutational, and structural evaluation of a Taxus phenylalanine aminomutase.

The structure of a phenylalanine aminomutase (TcPAM) from Taxus canadensis has been determined at 2.4 Å resolution. The active site of the TcPAM contains the signature 4-methylidene-1H-imidazol-5(4H)-one prosthesis, observed in all catalysts of the class I lyase-like family. This catalyst isomerizes (S)-α-phenylalanine to the (R)-ß-isomer by exchange of the NH2/H pair. The stereochemistry of the TcPAM reaction product is opposite of the (S)-ß-tyrosine made by the mechanistically related tyrosine aminomutase (SgTAM) from Streptomyces globisporus. Since TcPAM and SgTAM share similar tertiary- and quaternary-structures and have several highly conserved aliphatic residues positioned analogously in their active sites for substrate recognition, the divergent product stereochemistries of these catalysts likely cannot be explained by differences in active site architecture. The active site of the TcPAM structure also is in complex with (E)-cinnamate; the latter functions as both a substrate and an intermediate. To account for the distinct (3R)-ß-amino acid stereochemistry catalyzed by TcPAM, the cinnamate skeleton must rotate the C1-Cα and Cipso-Cß bonds 180° in the active site prior to exchange and rebinding of the NH2/H pair to the cinnamate, an event that is not required for the corresponding acrylate intermediate in the SgTAM reaction. Moreover, the aromatic ring of the intermediate makes only one direct hydrophobic interaction with Leu-104. A L104A mutant of TcPAM demonstrated an ∼1.5-fold increase in kcat and a decrease in KM values for sterically demanding 3'-methyl-α-phenylalanine and styryl-α-alanine substrates, compared to the kinetic parameters for TcPAM. These parameters did not change significantly for the mutant with 4'-methyl-α-phenylalanine compared to those for TcPAM.

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)-ß-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(ß), respectively, of the substrate are removed and interchanged completely intramolecularly with inversion of configuration at the migration centers to form ß-phenylalanine. This is a contrast to the retention of configuration mechanism followed by TcPAM.

Semibiocatalytic Approach toward Regioisomerically Enriched Ethyl Dimethylpyrazines Important in Flavor Industries.

Alkylpyrazines are important heterocyclic compounds used as flavorants in food and beverage industries. In this study, a regioselective semibiocatalytic process was developed to synthesize 2-ethyl-3,5-dimethylpyrazine (235-EDMP) over its 3-ethyl-2,5-dimethyl pyrazine (325-EDMP) isomer and vice versa. We initially explored how sterics could direct the coupling orientations between diamines and diketones to access 235- or 325-EDMP selectively. Also, the physical parameters of the reaction conditions were changed, such as reduced temperature, the order-of-addition of the reactants, and supplementation with chiral zeolites to template the orientation of the coupling partners to direct the reaction regiochemistry. Each reaction trial resulted in 50:50 mixtures of the EDMP isomers. An alternative approach was explored to control the regioselectivity of the reactions; α-hydroxy ketones replaced the diketones as the electrophilic coupling reactant used in previous trial experiments. The hydroxy ketone reactants were made biocatalytically with pyruvate decarboxylase. The coupling reaction between 2-hydroxypentan-3-one and propane-1,2-diamine resulted in the desired 235-EDMP at >70% (∼77 mg) relative to 325-EDMP in the mixture. The 3-hydroxypentan-2-one congener was biocatalyzed and reacted with propane-1,2-diamine as a proof of principle to synthesize 325-EDMP (∼60% relative abundance, ∼73 mg) over 235-EDMP. These results suggested a mechanism that was directed by the hydroxy ketone electrophilicity and the sterics at the diamine nucleophilic centers.

Point mutations (Q19P and N23K) increase the operational solubility of a 2alpha-O-benzoyltransferase that conveys various acyl groups from CoA to a taxane acceptor.

Two site-directed mutations within the wild-type 2-O-benzoyltransferase (tbt) cDNA, from Taxus cuspidata plants, yielded an encoded protein containing replacement amino acids at Q19P and N23K that map to a solvent-exposed loop region. The likely significant changes in the biophysical properties invoked by these mutations caused the overexpressed, modified TBT (mTBT) to partition into the soluble enzyme fraction about 5-fold greater than the wild-type enzyme. Sufficient protein could now be acquired to examine the scope of the substrate specificity of mTBT by incubation with 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III that was mixed individually with various substituted benzoyls, alkanoyls, and (E)-butenoyl CoA donors. The mTBT catalyzed the conversion of each 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III to several 7,13-O,O-diacetyl-2-O-acyl-2-O-debenzoylbaccatin III analogues. The relative catalytic efficiency of mTBT with the 7,13-O,O-diacetyl-2-O-debenzoyl surrogate substrate and heterole carbonyl CoA substrates was slightly greater than with the natural aroyl substrate benzoyl CoA, while substituted benzoyl CoA thioesters were less productive. Short-chain hydrocarbon carbonyl and cyclohexanoyl CoA thioesters were also productive, where C(4) substrates were transferred by mTBT with approximately 10- to 17-fold greater catalytic efficiency compared to the transfer of benzoyl. The described broad specificity of mTBT suggests that a plethora of 2-O-acyl variants of the antimitotic paclitaxel can be assembled through biocatalytic sequences.

Separation of alpha- from beta-arylalanines by nickel nitrilotriacetate chromatography.

A method is described to separate alpha- from beta-arylalanines by ligand exchange chromatography on a nickel nitrilotriacetate agarose column with UV monitoring of the effluent. Separate mixtures containing an alpha- and beta-arylalanine pair (1 mg of each) were individually loaded onto the nickel resin pre-equilibrated with the mobile phase at room temperature, and the amino acids were eluted from the column with a gradient from pH 12.0-8.0. The beta-arylalanines eluted first, followed by the alpha-isomers. The four alpha/beta-amino acid pairs tested were well separated with baseline resolution. An aliquot of each fraction was chemically treated to derivatize the amino acids to their N-acyl methyl ester analogs, and their identities were confirmed by GC/MS analysis. The sample recovery was quantitative (>98%), and the column matrix was very resilient, as demonstrated by consistent separation of the solutes after approximately 100 preparative cycles.

An N-aroyltransferase of the BAHD superfamily has broad aroyl CoA specificity in vitro with analogues of N-dearoylpaclitaxel.

The native N-debenzoyl-2'-deoxypaclitaxel:N-benzoyltransferase (NDTBT), from Taxus plants, transfers a benzoyl group from the corresponding CoA thioester to the amino group of the beta-phenylalanine side chain of N-debenzoyl-2'-deoxypaclitaxel, which is purportedly on the paclitaxel (Taxol) biosynthetic pathway. To elucidate the substrate specificity of NDTBT overexpressed in Escherichia coli, the purified enzyme was incubated with semisynthetically derived N-debenzoyltaxoid substrates and aroyl CoA donors (benzoyl; ortho-, meta-, and para-substituted benzoyls; various heterole carbonyls; alkanoyls; and butenoyl), which were obtained from commercial sources or synthesized via a mixed anhydride method. Several unnatural N-aroyl-N-debenzoyl-2'-deoxypaclitaxel analogues were biocatalytically assembled with catalytic efficiencies (V(max)/K(M)) ranging between 0.15 and 1.74 nmol.min(-1).mM(-1). In addition, several N-acyl-N-debenzoylpaclitaxel variants were biosynthesized when N-debenzoylpaclitaxel and N-de(tert-butoxycarbonyl)docetaxel (i.e., 10-deacetyl-N-debenzoylpaclitaxel) were used as substrates. The relative velocity (v(rel)) for NDTBT with the latter two N-debenzoyl taxane substrates ranged between approximately 1% and 200% for the array of aroyl CoAs compared to benzoyl CoA. Interestingly, NDTBT transferred hexanoyl, acetyl, and butyryl more rapidly than butenoyl or benzoyl from the CoA donor to taxanes with isoserinoyl side chains, whereas N-debenzoyl-2'-deoxypaclitaxel was more rapidly converted to its N-benzoyl derivative than to its N-alkanoyl or N-butenoyl congeners. Biocatalytic N-acyl transfer of novel acyl groups to the amino functional group of N-debenzoylpaclitaxel and its 2'-deoxy precursor reveal the surprisingly indiscriminate specificity of this transferase. This feature of NDTBT potentially provides a tool for alternative biocatalytic N-aroylation/alkanoylation to construct next generation taxanes or other novel bioactive diterpene compounds.