Publications

• Aristolochene synthase: purification, molecular cloning, high-level expression in Escherichia coli, and characterization of the Aspergillus terreus cyclase.

  Cane D.E., Kang I.

  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 dege ... >> More

  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. << Less

  Arch. Biochem. Biophys. 376:354-364(2000) [PubMed] [EuropePMC]

• Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase.

  Starks C.M., Back K., Chappell J., Noel J.P.

  Terpene cyclases catalyze the synthesis of cyclic terpenes with 10-, 15-, and 20-carbon acyclic isoprenoid diphosphates as substrates. Plants have been a source of these natural products by providing a homologous set of terpene synthases. The crystal structures of 5-epi-aristolochene synthase, a s ... >> More

  Terpene cyclases catalyze the synthesis of cyclic terpenes with 10-, 15-, and 20-carbon acyclic isoprenoid diphosphates as substrates. Plants have been a source of these natural products by providing a homologous set of terpene synthases. The crystal structures of 5-epi-aristolochene synthase, a sesquiterpene cyclase from tobacco, alone and complexed separately with two farnesyl diphosphate analogs were analyzed. These structures reveal an unexpected enzymatic mechanism for the synthesis of the bicyclic product, 5-epi-aristolochene, and provide a basis for understanding the stereochemical selectivity displayed by other cyclases in the biosynthesis of pharmacologically important cyclic terpenes. As such, these structures provide templates for the engineering of novel terpene cyclases. << Less

  Science 277:1815-1820(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Crystal structure determination of aristolochene synthase from the blue cheese mold, Penicillium roqueforti.

  Caruthers J.M., Kang I., Rynkiewicz M.J., Cane D.E., Christianson D.W.

  The 2.5-A resolution crystal structure of recombinant aristolochene synthase from the blue cheese mold, Penicillium roqueforti, is the first of a fungal terpenoid cyclase. The structure of the enzyme reveals active site features that participate in the cyclization of the universal sesquiterpene cy ... >> More

  The 2.5-A resolution crystal structure of recombinant aristolochene synthase from the blue cheese mold, Penicillium roqueforti, is the first of a fungal terpenoid cyclase. The structure of the enzyme reveals active site features that participate in the cyclization of the universal sesquiterpene cyclase substrate, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. Metal-triggered carbocation formation initiates the cyclization cascade, which proceeds through multiple complex intermediates to yield one exclusive structural and stereochemical isomer of aristolochene. Structural homology of this fungal cyclase with plant and bacterial terpenoid cyclases, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of terpene biosynthesis. << Less

  J. Biol. Chem. 275:25533-25539(2000) [PubMed] [EuropePMC]

• Germacrene A is a product of the aristolochene synthase-mediated conversion of farnesylpyrophosphate to aristolochene.

  Calvert M.J., Ashton P.R., Allemann R.K.

  The biosynthesis of several sesquiterpenes has been proposed to proceed via germacrene A. However, to date, the production of germacrene A has not been proven directly for any of the sesquiterpene synthases for which it was postulated as an intermediate. We demonstrate here for the first time that ... >> More

  The biosynthesis of several sesquiterpenes has been proposed to proceed via germacrene A. However, to date, the production of germacrene A has not been proven directly for any of the sesquiterpene synthases for which it was postulated as an intermediate. We demonstrate here for the first time that significant amounts of germacrene A (7.5% of the total amount of products) are indeed released from wild-type aristolochene synthase (AS) from Penicillium roqueforti. Germacrene A was identified through direct GC-MS comparison to an authentic sample and through production of beta-elemene in a thermal Cope rearrangement. AS also produced a small amount of valencene through deprotonation of C6 rather than C8 in the final step of the reaction. On the basis of the X-ray structure of AS, Tyr 92 was postulated to be the active-site acid responsible for protonation of germacrene A (Caruthers, J. M.; Kang, I.; Rynkiewicz, M. J.; Cane, D. E.; Christianson, D. W. J. Biol. Chem. 2000, 275, 25533-25539). The CD spectra of a mutant protein, ASY92F, in which Tyr 92 was replaced by Phe, and of AS were very similar. ASY92F was approximately 0.1% as active as nonmutated recombinant AS. The steady-state kinetic parameters were measured as 0.138 min(-1) and 0.189 mM for k(cat) and K(M), respectively. Similar to a mutant protein of 5-epi-aristolochene (Rising, K. A.; Starks, C. M.; Noel, J. P.; Chappell, J. J. Am. Chem. Soc. 2000, 122, 1861-1866), the mutant released significant amounts of germacrene A (approximately 29%). ASY92F also produced various amounts of a further five hydrocarbons of molecular weight 204, valencene, beta-(E)-farnesene, alpha- and beta-selinene, and selina-4,11-diene. << Less

  J Am Chem Soc 124:11636-11641(2002) [PubMed] [EuropePMC]

• Aristolochene synthase. Isolation, characterization, and bacterial expression of a sesquiterpenoid biosynthetic gene (Ari1) from Penicillium roqueforti.

  Proctor R.H., Hohn T.M.

  Aristolochene is the likely precursor of the sesquiterpenoid toxins produced by a number of filamentous fungi. One of these, PR-toxin, is produced by Penicillium roqueforti. We report here the isolation of a gene (Ari1) coding for the sesquiterpene cyclase, aristolochene synthase (AS), from P. roq ... >> More

  Aristolochene is the likely precursor of the sesquiterpenoid toxins produced by a number of filamentous fungi. One of these, PR-toxin, is produced by Penicillium roqueforti. We report here the isolation of a gene (Ari1) coding for the sesquiterpene cyclase, aristolochene synthase (AS), from P. roqueforti. Nucleotide sequence analysis of genomic and cDNA clones revealed that the Ari1 gene contains two introns. A Protein A/AS fusion enzyme was expressed in Escherichia coli and shown to have sesquiterpene cyclase activity. Analysis of the Protein A/AS fusion enzyme reaction mixtures by TLC and gas chromatography/mass spectrometry identified aristolochene as a major product. The Ari1 gene encodes a polypeptide of molecular weight 39,200. Expression of Ari1 occurs in stationary phase cultures of P. roqueforti and appears to be transcriptionally regulated. << Less

  J. Biol. Chem. 268:4543-4548(1993) [PubMed] [EuropePMC]