Molecular Cloning and Characterization of the Phenylalanine Aminomutase Gene From Taxus baccata L.

Abstract

Background: Taxol is one of the most important anti-cancer drugs, which is obtained from yew trees (Taxus sp.). T+he first step in side chain assembly of taxol is catalyzed by phenylalanine aminomutase, which converts α-phenylalanine to β-phenylalanine. Objectives: In this study, for the first time, we report on the cloning, preliminary expression and characterization of a full-length gene and cDNA encoding phenylalanine aminomutase from Taxus baccata L. Materials and Methods:Comparison of the full-length gene with other ones identified from the Taxus species showed high similarity, particularly with Taxus x media. Results:The results showed that the expression level of this gene in Taxus baccata is very low and therefore this enzymatic step could be a rate limiting step in the taxol biosynthesis pathway. Successful amplification of the cDNA was only obtained from RNA samples isolated from methyl jasmonate elicited suspension cells of Taxus baccata. The cloned cDNA contained a 2064 bp open reading frame encoding a protein composed of 687 amino acids. Sequence comparison analysis revealed that the gene is very similar (98 -99 %) with respect to the nucleotide and amino acid sequences in different Taxus species and also share the signature active site motif (175ASG177). Conclusions: The predicted structure of TbPAM was analyzed using bioinformatic tools. The results indicated that the protein has similar overall folding to tyrosine aminomutase.