Disease relevance of aristolochene

• The aristolochene synthase gene (Ari1) of P. roqueforti has previously been cloned and expressed at low levels as a protein A-aristolochene synthase fusion protein in Escherichia coli [1].

High impact information on 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 [2].
• Aristolochene synthase: mechanistic analysis of active site residues by site-directed mutagenesis [3].
• Incubation of farnesyl diphosphate (1) with Penicillium roqueforti aristolochene synthase yielded (+)-aristolochene (4), accompanied by minor quantities of the proposed intermediate (S)-(-)germacrene A (2) and the side-product (-)-valencene (5) in a 94:4:2 ratio [3].
• 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 [4].
• Dual role for phenylalanine 178 during catalysis by aristolochene synthase [5].

Chemical compound and disease context of aristolochene

• 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 [6].

Biological context of aristolochene

• 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 [6].
• 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 [6].
• 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 [6].

Anatomical context of aristolochene

• We have now used the polymerase chain reaction to amplify the aristolochene synthase coding sequence using engineered primers which produced dsDNA carrying an EcoRI restriction site and the T7 gene 10 ribosome binding site and translational spacer element immediately upstream of the ATG start codon and a BamHI site adjacent to the TAA stop codon [1].

Associations of aristolochene with other chemical compounds

• The mechanistic details of the cyclisation of farnesylpyrophosphate (FPP) by aristolochene synthase (AS) from Penicillium roqueforti have only recently begun to emerge, mainly through the analysis of the reaction products generated by AS-mutants [7].

Gene context of aristolochene

• A mutant of Aristolochene Synthase (AS), in which Tyr 92 was replaced by Val, produced the alicyclic beta-(E)-farnesene as the major product, indicating that cyclisation of FPP is controlled by Tyr 92 in AS [8].

Analytical, diagnostic and therapeutic context of aristolochene

• Analysis of the Protein A/AS fusion enzyme reaction mixtures by TLC and gas chromatography/mass spectrometry identified aristolochene as a major product [9].
• Site-directed mutagenesis of amino acid residues in two highly conserved Mg(2+)-binding domains led in most cases to reductions in both k(cat) and k(cat)/K(m) as well as increases in the proportion of (S)-(-)germacrene A (2), with the E252Q mutant of the P. roqueforti aristolochene synthase producing only (-)-2 [3].
• PCR was used to insert the A. terreus aristolochene synthase gene into the T7lac expression vector pET11a [6].
• The sesquiterpene cyclase, aristolochene synthase, has been purified from Penicillium roqueforti by gel filtration and anion-exchange chromatography [10].
• Aristolochene synthase: purification, molecular cloning, high-level expression in Escherichia coli, and characterization of the Aspergillus terreus cyclase [6].

References