Abstract
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.
Highlights
Aristolochene synthase is a terpenoid cyclase from the blue cheese mold, Penicillium roqueforti, that catalyzes the metal-dependent cyclization of farnesyl diphosphate to form the bicyclic hydrocarbon aristolochene (Fig. 1) [1]
The atomic coordinates of native aristolochene synthase have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ with accession code 1DI1
The atomic coordinates of the models of aristolochene synthase complexed with farnesyl diphosphate, germacrene A, eudesmane cation, and aristolochene have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics with accession codes 1F1P, 1F1L, 1F1N, and 1F1K, respectively
Summary
Expression and Purification of Aristolochene Synthase—From a glycerol stock of E. coli BL21(DE3) carrying pZW04, 5 ml of seed culture in LBampicillin was cultured at 37 °C overnight. 500 ml of prewarmed (30 °C) LB-ampicillin media in a 3-liter flask was inoculated with this overnight seed, giving an initial A at 600 nm of 0.05, and further incubated at 30 °C until the A reached 0.7–1.0. Expression and Purification of Aristolochene Synthase—From a glycerol stock of E. coli BL21(DE3) carrying pZW04, 5 ml of seed culture in LBampicillin was cultured at 37 °C overnight. 500 ml of prewarmed (30 °C) LB-ampicillin media in a 3-liter flask was inoculated with this overnight seed, giving an initial A at 600 nm of 0.05, and further incubated at 30 °C until the A reached 0.7–1.0. Isopropyl-1-thio--D-galactopyranoside was added to 0.5 mM final concentration to induce production of recombinant aristolochene synthase. Cells were harvested after a 4-h induction period, resuspended in 50 ml of cell lysis buffer (5 mM EDTA, 5 mM -mercaptoethanol, 20 mM Tris (pH 7.5)), and sonicated to disrupt cells.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
