Cloning, heterologous expression, and characterization of a phenylalanine aminomutase involved in Taxol biosynthesis.

Biosynthesis of the N-benzoyl phenylisoserinoyl side chain of the anticancer drug Taxol starts with the conversion of 2S-alpha-phenylalanine to 3R-beta-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 chinensis, acquired recently by a reverse genetics approach (Bristol-Myers Squibb (August 14, 2003) U. S. Patent WO 03/066871 A2). The full-length cDNA has an open reading frame of 2094 base pairs and encodes a protein of 698 residues with a calculated molecular mass of 76,530 Da. The recombinant mutase has a pH optimum of 8.5, a k(cat) value of 0.015 s(-1), and a K(m) of 45 +/- 8 microm for 2S-alpha-phenylalanine. The stereochemical mechanism of PAM involves the removal and interchange of the pro-3S hydrogen and the amino group, which rebonds at C-3 with retention of configuration. The recombinant enzyme appears to catalyze both the forward and reverse reactions with specificity for both 2S-alpha-phenylalanine and 3S- or 3R-beta-phenylalanine substrates, respectively, whereas the related phenylpropanoids 2S-aminocyclohexanepropanoic acid, 2R-alpha-phenylalanine, and 2S-alpha-tyrosine are not converted to their beta-isomers by the mutase.