Biosynthesis of the N-benzoyl phenylisoserinoyl side chain of the anticancer drug Taxol starts with the conversion of 2S-␣-phenylalanine to 3R-␤-phenylalanine by phenylalanine aminomutase (PAM). A gene cloning approach was based on the assumption that PAM would resemble the well known plant enzyme phenylalanine ammonia lyase. A phenylalanine ammonia lyase-like sequence acquired from a Taxus cuspidata cDNA library was expressed functionally in Escherichia coli and confirmed as the target aminomutase that is virtually identical to the recombinant enzyme and clone from Taxus The final stages of Taxol biosynthesis (see Fig. 1A) in yew species involve the assembly and attachment to C-13 of the taxane core of the N-benzoyl phenylisoserinoyl side chain, which is an important pharmacophoric descriptor of this anticancer drug (1, 2). In the current practice of Taxol production, this side chain is attached by chemical semisynthesis to baccatin III, which is derived from 10-deacetylbaccatin III, a Taxus (yew) metabolite that is much more readily available than Taxol itself (3-5). In the biosynthetic pathway, five steps are involved in the construction of the side chain. The first step is considered to be the conversion of 2S-␣-phenylalanine to 3R-␤-phenylalanine by an aminomutase that catalyzes an intramolecular migration of the amino group and a partial internal transfer of the pro-3S hydrogen (6, 7). This step is seemingly followed by the ligase-mediated activation to the corresponding CoA ester and then the transfer of ␤-phenylalanoyl to the C-13 hydroxyl of baccatin III. The resulting intermediate (designated ␤-phenylalanoyl baccatin III or N-debenzoyl-2Ј-deoxytaxol) then likely undergoes cytochrome P450-mediated hydroxylation at the side chain 2Ј-position to generate the isoserinoyl moiety and final N-benzoylation of this side chain (8) to complete the biosynthesis of Taxol. cDNAs encoding the two transferases involved in C-13 side chain assembly have been described previously (8,9).The relative abundance of ␤-phenylalanine and side chaindeficient late pathway metabolites such as baccatin III and 10-deacetylbaccatin III in vivo (10) suggests that either the CoA ligase for ␤-phenylalanine or the CoA ester-dependent ␤-phenylalanoyltransferase (9), both of which function downstream of the aminomutase, may be rate limiting in side chain assembly and, thus, in Taxol biosynthesis. Because the phenylalanine aminomutase (PAM) 1 catalyzes the first step of the side chain assembly process and shares its primary metabolite substrate, phenylalanine, with several competing, non-taxoid phenylpropanoid pathway enzymes in plants (11-13), it is therefore an important target for genetic engineering in yew or derived cell cultures to increase Taxol production yields. PAM is also of interest enzymologically because the reaction that is catalyzed is unusual, and, in addition to the adenosylcobalamin-dependent leucine 2,3-aminomutase from Andrographis paniculata and potato tubers (14 -16), it is the only other aminomutase of plant origin d...

The phenylalanine aminomutase from Taxus catalyzes the vicinal exchange of the amino group and the pro-3S hydrogen of (2S)-alpha-phenylalanine to make (3R)-beta-phenylalanine. While the migration of the amino group from C2 of the substrate to C3 of the product is already known to proceed intramolecularly with retention of configuration, the stereochemistry of the hydrogen transfer remained unknown, until now. The chemical shifts of the prochiral hydrogens of authentic (3R)-beta-phenylalanine were established by 1H NMR, and the configuration of each hydrogen was assigned by 2H NMR analysis of a racemic mixture of [2,3-2H2]-(2S,3R)- and (2R,3S)-beta-phenylalanines synthesized via syn addition of deuterium gas with palladium catalyst to stereospecifically reduce the double bond of an N-acetyl enamine. After the aminomutase was incubated with [3,3-2H2]-(2S)-alpha-phenylalanine, the derived deuterium-labeled beta-diastereoisomer product, derivatized as the N-acetyl methyl ester, was analyzed by 2H NMR, which revealed that the mutase shuttles the pro-3S hydrogen to C2 of the beta-isomer product (designated 2S,3R) with retention of configuration. Retention of configuration at both reaction termini is unique among all aminomutase mechanisms examined so far. Furthermore, the dynamics of the Cbeta-H bond of the substrate were measured in a competitive experiment with deuterium-labeled substrate to calculate a primary kinetic isotope effect on Vmax/KM of 2.0 +/- 0.2, indicating that C-H bond cleavage is likely rate limiting. Isotope exchange data indicate that the migratory deuterium of [2H8]-(2S)-alpha-phenylalanine, at saturation, dynamically exchanges up to 75%, with protons from the solvent during the reaction after the first 10% of product is formed. The calculated equilibrium constant of 1.1 indicates that the beta-isomer was slightly favored relative to the alpha-isomer at 30 degrees C.

The substrate specificity of a Taxus-derived phenylalanine aminomutase (PAM) was investigated, and the enzyme was found to catalyze the conversion of variously substituted vinyl- and aryl-S-α-alanines to corresponding β-amino acids. This study shows the broad substrate specificity of PAM and thus demonstrates a potential, practical biosynthetic route toward unnatural β-amino acid subunits of Taxol analogues and β-peptides.

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