Molecular cloning of a taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli.
The taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase which catalyzes the third step of Taxol biosynthesis has been isolated from methyl jasmonate-induced Taxus cells, and partially purified and characterized (K. Walker, R. E. B. Ketchum, M. Hezari, D. Gatfield, M. Golenowski, A. Barthol, and R. Croteau, Arch. Biochem. Biophys. 364, 273-279 1999). A revised purification method allowed internal amino acid microsequencing of the enzyme, from which primers were designed and employed to amplify a transacetylase gene-specific fragment. This radiolabeled, 900-bp amplicon was used as a hybridization probe to screen a cDNA library constructed from poly(A)(+) RNA isolated from induced Taxus cells, from which a full-length transacetylase sequence was obtained. Expression of this clone from pCWori(+) in Escherichia coli JM109 cells yielded the functional enzyme, as determined by radiochemical assay and combined capillary gas chromatographic-mass spectrometric verification of the acetylated product. The full-length DNA has an open-reading frame of 1317 nucleotides corresponding to a deduced amino acid sequence of 439 residues that exhibits high sequence identity to the proteolytic fragments of the native enzyme, which the recombinant transacetylase resembles in properties. Consistent with the size of the operationally soluble native enzyme, the DNA appears to encode a monomeric protein of molecular weight 49,079 that bears no N-terminal organellar targeting information. Sequence comparison of the taxadien-5alpha-ol-O-acetyl transferase with the few other known acyl transferases of plant origin indicates a significant degree of similarity between these enzymes (64-67%). The efficient conversion of taxadien-5alpha-yl acetate to further hydroxylated intermediates of the Taxol pathway confirms the significance of this acylation step and suggests this taxadienol transacetylase to be an important target for genetic manipulation to improve Taxol production.
Walker
K
K
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
Schoendorf
A
A
Croteau
R
R
D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D008401:Gas Chromatography-Mass Spectrometry; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010946:Plants, Medicinal; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020946:Taxus / Q000201:enzymology* / Q000235:genetics; D014462:Ultrafiltration
D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000123:Acetyltransferases; C405086:taxa-4(20),11(12)-dien-5alpha-ol-O-acetyltransferase