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Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis.

ABSTRACT: The structural pharmacophore of Taxol, responsible for binding the N terminus of the beta-subunit of tubulin to arrest cell proliferation, comprises, in part, the 13-O-(N-benzoyl-3-phenylisoserinoyl) side chain. To identify the side chain transferase of Taxol biosynthesis, a set of transacylases obtained from an enriched cDNA library (constructed from mRNA isolated from Taxus cuspidata cells induced with methyl jasmonate for Taxol production) was screened. A cDNA clone (designated TAX7) encoding a taxoid C-13 O-phenylpropanoyltransferase was isolated which yielded a recombinant enzyme that catalyzes the selective 13-O-acylation of baccatin III with beta-phenylalanoyl CoA as the acyl donor to form N-debenzoyl-2'-deoxytaxol. This enzymatic product was converted to 2'-deoxytaxol by chemical N-benzoylation, and the identity of this derivative was confirmed by spectrometric analyses. The full-length cDNA has an ORF of 1,335 bases and encodes a 445-aa protein with a calculated molecular weight of 50,546. Evaluation of kinetic parameters revealed K(m) values of 2.4 +/- 0.5 microM and 4.9 +/- 0.3 microM for baccatin III and beta-phenylalanoyl-CoA, respectively. The pH optimum for the recombinant O-(3-amino-3-phenylpropanoyl)transferase is at 6.8. Identification of this clone completes acquisition of the five aroyl/acyltransferases involved in the biosynthesis of Taxol. Application of these transacylase genes in suitable host cells can improve the production yields of Taxol and could enable the preparation of second-generation Taxol analogs possessing greater bioactivity and improved water solubility.

SUBMITTER: Walker K

PROVIDER: S-EPMC130526 | biostudies-literature | 2002 Oct

REPOSITORIES: biostudies-literature

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Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis.

Walker Kevin K Fujisaki Shingo S Long Robert R Croteau Rodney R

Proceedings of the National Academy of Sciences of the United States of America 20020913 20

The structural pharmacophore of Taxol, responsible for binding the N terminus of the beta-subunit of tubulin to arrest cell proliferation, comprises, in part, the 13-O-(N-benzoyl-3-phenylisoserinoyl) side chain. To identify the side chain transferase of Taxol biosynthesis, a set of transacylases obtained from an enriched cDNA library (constructed from mRNA isolated from Taxus cuspidata cells induced with methyl jasmonate for Taxol production) was screened. A cDNA clone (designated TAX7) encoding ...[more]

PMID: 12232048

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Project description:BackgroundTriacylglycerides (TAGs) are a class of neutral lipids that represent the most important storage form of energy for eukaryotic cells. DGAT (acyl-CoA: diacylglycerol acyltransferase; EC 2.3.1.20) is a transmembrane enzyme that acts in the final and committed step of TAG synthesis, and it has been proposed to be the rate-limiting enzyme in plant storage lipid accumulation. In fact, two different enzymes identified in several eukaryotic species, DGAT1 and DGAT2, are the main enzymes responsible for TAG synthesis. These enzymes do not share high DNA or protein sequence similarities, and it has been suggested that they play non-redundant roles in different tissues and in some species in TAG synthesis. Despite a number of previous studies on the DGAT1 and DGAT2 genes, which have emphasized their importance as potential obesity treatment targets to increase triacylglycerol accumulation, little is known about their evolutionary timeline in eukaryotes. The goal of this study was to examine the evolutionary relationship of the DGAT1 and DGAT2 genes across eukaryotic organisms in order to infer their origin.ResultsWe have conducted a broad survey of fully sequenced genomes, including representatives of Amoebozoa, yeasts, fungi, algae, musses, plants, vertebrate and invertebrate species, for the presence of DGAT1 and DGAT2 gene homologs. We found that the DGAT1 and DGAT2 genes are nearly ubiquitous in eukaryotes and are readily identifiable in all the major eukaryotic groups and genomes examined. Phylogenetic analyses of the DGAT1 and DGAT2 amino acid sequences revealed evolutionary partitioning of the DGAT protein family into two major DGAT1 and DGAT2 clades. Protein secondary structure and hydrophobic-transmembrane analysis also showed differences between these enzymes. The analysis also revealed that the MGAT2 and AWAT genes may have arisen from DGAT2 duplication events.ConclusionsIn this study, we identified several DGAT1 and DGAT2 homologs in eukaryote taxa. Overall, the data show that DGAT1 and DGAT2 are present in most eukaryotic organisms and belong to two different gene families. The phylogenetic and evolutionary analyses revealed that DGAT1 and DGAT2 evolved separately, with functional convergence, despite their wide molecular and structural divergence.

Dataset Information

Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis.

Publications

Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis.

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