Aristolochene synthase: purification, molecular cloning, high-level expression in Escherichia coli, and characterization of the Aspergillus terreus cyclase.
Aristolochene synthase catalyzes the cyclization of farnesyl diphosphate (6) to (+)-aristolochene (1). The Aspergillus terreus enzyme has been purified 75-fold to homogeneity in six steps. Based on the sequence of 3 internal peptides obtained by Lys-C digestion of the native protein, a set of degenerate PCR primers was used to amplify a 550-bp segment of cDNA corresponding to a portion of the aristolochene synthase transcript. A second round of PCR using specific primers was used to prepare a (32)P-labeled 180-bp segment, which was used to screen an A. terreus cDNA library prepared using lambdaZapII, resulting in the identification and sequencing of the A. terreus aristolochene synthase cDNA. Aristolochene synthase was encoded by an open reading frame (ORF) of 960 bp, corresponding to a protein of 320 amino acids with a predicted M(D) of 36,480. Comparison of the A. terreus ORF with the sequence of the previously described aristolochene synthase from Penicillium roqueforti revealed a 66% of identity at the nucleic acid level and a 70% identity at the deduced amino acid level between the aristolochene synthases from the two different fungal sources. PCR was used to insert the A. terreus aristolochene synthase gene into the T7lac expression vector pET11a. Cloning of the resultant construct into Escherichia coli XL1-Blue and subcloning into the expression host E. coli BL21(DE3)/pLysS gave, after induction with IPTG, soluble aristolochene synthase as 5-10% of total protein. The recombinant aristolochene synthase, which was purified 13-fold to homogeneity, appeared to be identical in all respects with the native A. terreus enzyme, displaying essentially the same steady-state kinetic parameters, with a K(m) of 15 nM and k(cat) 0.015 s(-1). Using PCR to amplify the aristolochene synthase gene (Aril) from A. terreus genomic DNA revealed the presence of 2 introns, identical in relative location but different in both sequence and length compared to the corresponding Ari1 gene of P. roqueforti.
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Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA. david_cane@brown.edu
Kang
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D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal / Q000235:genetics; D007438:Introns / Q000235:genetics; D007535:Isomerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010407:Penicillium / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013816:Thermodynamics
D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C054277:aristolochene; D007535:Isomerases; C060050:aristolochene synthase