ABSTRACT
Paclitaxel® (PC) is one of the most effective and profitable anti-cancer drugs. The most promising sources of this compound are natural materials such as tissue cultures of Taxus species and, more recently, hazelnut (Corylus avellana L.). A large part of the PC biosynthetic pathway in the yew tree and a few steps in the hazelnut have been identified. Since understanding the biosynthetic pathway of plant-based medicinal metabolites is an effective step toward their development and engineering, this paper aimed to identify taxadiene-5α-ol-O-acetyltransferase (TDAT) in hazelnut. TDAT is one of the key genes involved in the third step of the PC biosynthetic pathway. In this study, the TDAT gene was isolated using the nested-PCR method and then characterized. The cotyledon-derived cell mass induced with 150 µM of methyl jasmonate (MeJA) was utilized to isolate RNA and synthesize the first-strand cDNA. The full-length cDNA of TDAT is 1423 bp long and contains a 1302 bp ORF encoding 433 amino acids. The phylogenetic analysis of this gene revealed high homology with its ortholog genes in Quercus suber and Juglans regia. Bioinformatics analyses were used to predict the secondary and tertiary structures of the protein. Due to the lack of signal peptide, protein structure prediction suggested that this protein may operate at the cytoplasm. The homologous superfamily of the T5AT protein, encoded by TDAT, has two domains. The highest and lowest hydrophobicity of amino acids were found in proline 142 and lysine 56, respectively. T5AT protein fragment had 24 hydrophobic regions. The tertiary structure of this protein was designed using Modeler software (V.9.20), and its structure was verified based on the results of the Verify3D (89.46%) and ERRAT (90.3061) programs. The T5AT enzyme belongs to the superfamily of the transferase, and the amino acids histidine 164, cysteine 165, leucine 166, histidine 167, and Aspartic acid 168 resided at its active site. More characteristics of TDAT, which would aid PC engineering programs and maximize its production in hazelnut, were discussed.
