28028-64-0Relevant articles and documents
(+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.
Prosser, Ian,Phillips, Andy L,Gittings, Simon,Lewis, Mervyn J,Hooper, Antony M,Pickett, John A,Beale, Michael H
, p. 691 - 702 (2002)
Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.
Genome mining in streptomyces avermitilis: Cloning and characterization of sav-76, the synthase for a new sesquiterpene, avermitilol
Chou, Wayne K. W.,Fanizza, Immacolata,Uchiyama, Takuma,Komatsu, Mamoru,Ikeda, Haruo,Cane, David E.
supporting information; experimental part, p. 8850 - 8851 (2010/08/21)
The terpene synthase encoded by the sav76 gene of Streptomyces avermtilis was expressed in Escherichia coli as an N-terminal-His6-tag protein, using a codon-optimized synthetic gene. Incubation of the recombinant protein, SAV-76, with farnesyl diphosphate (1, FPP) in the presence of Mg2+ gave a new sesquiterpene alcohol avermitilol (2), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR, along with minor amounts of germacrene A (3), germacrene B (4), and viridiflorol (5). The absolute configuration of 2 was assigned by 1H NMR analysis of the corresponding (R)- and (S)-Mosher esters. The steady state kinetic parameters were kcat 0.040 ± 0.001 s-1 and Km 1.06 ± 0.11 μM. Individual incubations of recombinant avermitilol synthase with [1,1-2H2]FPP (1a), (1S)-[1-2H]-FPP (1b), and (1R)-[1-2H]-FPP (1c) and NMR analysis of the resulting avermitilols supported a cyclization mechanism involving the loss of H-1 re to generate the intermediate bicyclogermacrene (7), which then undergoes proton-initiated anti-Markovnikov cyclization and capture of water to generate 2. A copy of the sav76 gene was reintroduced into S. avermitilis SUKA17, a large deletion mutant from which the genes for the major endogenous secondary metabolites had been removed, and expressed under control of the native S. avermitilis promoter rpsJp (sav4925). The resultant transformants generated avermitilol (2) as well as the derived ketone, avermitilone (8), along with small amounts of 3, 4, and 5. The biochemical function of all four terpene synthases found in the S. avermtilis genome have now been determined.
Aristolochene synthase: Mechanistic analysis of active site residues by site-directed mutagenesis
Felicetti, Brunella,Cane, David E.
, p. 7212 - 7221 (2007/10/03)
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. By contrast, the closely related aristolochene synthase from Aspergillus terreus cyclized farnesyl diphosphate only to (+)-aristolochene (4). Site-directed mutagenesis of amino acid residues in two highly conserved Mg2+-binding domains led in most cases to reductions in both kcat and kcat/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. The P. roqueforti D115N, N244L, and S248A/E252D mutants were inactive, as was the A. terreus mutant E227Q. The P. roqueforti mutant Y92F displayed a 100-fold reduction in kcat that was offset by a 50-fold decrease in Km, resulting in a relatively minor 2-fold decrease in catalytic efficiency, kcat/Km. The finding that Y92F produced (+)-aristolochene (4) as 81% of the product, accompanied by 7% 5 and 12% 2, rules out Tyr-92 as the active site Lewis acid that is responsible for protonation of the germacrene A intermediate in the formation of aristolochene (4).
