Purification, cloning, and functional expression of phenylalanine aminomutase: The first committed step in Taxol side-chain biosynthesis☆
Section snippets
Cell culture
Taxus chinensis cell suspensions were kindly provided by Phyton (Ithaca, NY). Cell cultures were maintained and grown as previously described [7]. Cell suspension was exchanged into Taxol production medium for 6 days before they were harvested and frozen as previously described [7].
PAM assay, purification, and characterization
All fine chemicals were purchased from either Sigma (St. Louis, MO) or VWR (New York, NY) unless otherwise indicated. A typical PAM assay mixture contains 1–10 μg enzyme in 50 mM Tris–HCl buffer (pH 7.5) with 5 mM α-l
Purification and characterization of PAM from T. chinensis
Crude extracts of PAM were prepared from cultured plant cells via a four-step purification, resulting in more than 150-fold purification (Table 1). Reactive Green 19 chromatography was a key step for the purification scheme as this column separates PAM from PAL, an enzyme that utilizes the same α-phenylalanine substrate and has a similar molecular weight. The presence of Mg2+ was critical during Reactive Green 19 chromatography since PAL only binds to the column in the presence of Mg2+.
SDS–PAGE
Discussion
In this report, the novel enzyme catalyzing the first biosynthetic step leading to the Taxol side-chain, PAM has been purified to apparent homogeneity and thoroughly characterized. The cloned cDNA has been conclusively demonstrated to be the pam gene by presence of all eight-peptide sequences generated from the purified active PAM protein (Fig. 3) and the presence of PAM activity in extracts of E. coli containing the cloned pam cDNA (Fig. 4). The product of the recombinant PAM was demonstrated
Acknowledgments
We gratefully acknowledge Stephen Tindall (Argo BioAnalytica) for peptide sequencing effort, and the staff at Phyton, Inc. for providing the Taxus cell cultures.
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2021, Catalysis TodayCitation Excerpt :It is an excellent choice to utilize the benefits of biocatalysis in production of enantiopure API molecules. Enantiopure aromatic α- and β-amino acids are frequently occurring building blocks of natural secondary metabolites such as andrimide [4], enedyine C-1027 [5], taxol [6,7], chondramide C, l-DOPA [8] or l-thyroxine. Thus, enantiopure α- and β-amino acids and their unnatural analogues are well-applicable precursors of nuclear hormone inhibitors, hepatitis C antiviral agents or cholecystokinin B antagonist and are frequently used as building blocks to create novel peptide or cyclopeptide APIs [9,10].
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Data deposition: The sequence reported in this paper has been deposited in the GenBank database (Accession No. AY724735).
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Present address: Enzyme Technology Development, Process Research and Development, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 08903, USA.
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Present address: Nereus Pharmaceuticals, Inc., 10480 Wateridge Circle, San Diego, CA 92121, USA.