Characterization and molecular cloning of a sesquiterpene cyclase: Artistolochene synthase from Aspergillus terreus

Aristolochene synthase (ASAT), a sesquiterpene cyclase, was purified from the native source, the fungus Aspergillus terreus, by employing standard protein purification techniques including ion exchange chromatography, hydrophobic interaction chromatography and gel filtration chromatography.;Internal peptide sequences of the purified protein were obtained after specific proteolytic digestion, and based on these sequences several PCR primers were designed. PCR was carried out using these primers resulting in amplification of a partial ASAT gene. The amplified DNA product was cloned and sequenced. A cDNA library, constructed from A. terreus mRNA was screened for the ASAT gene using a radio-labeled probe based on the partial gene sequence. One full length ORF of 960 bp (encoding a 320 amino acid protein) was identified and overexpressed in Escherichia coli host. The ASAT enzyme contained an aspartate-rich motif that is common to all known prenyl transferases and terpene cyclases.;Both native and recombinant ASAT were characterized. The Km and kcat of both enzymes were 14 nM and 0.11 s -1, respectively.;Genomic DNA encoding ASAT was also cloned by PCR. It contained two introns whose locations were identical to those of genomic DNA of ASPR, the aristolochene synthase from Penicillium roqueforti. The similarity and identity of ASAT to ASPR at the protein level were 70% and 63%, respectively and the identity at the DNA level was 67%, indicating that these two enzymes are closely related.;ASAT was inducible when the cultures of A. terreus were subjected to cold shock, suggesting that this enzyme may be involved in defense mechanisms when culture conditions are sub-optimal.