Biosynthesis of aromatic isoprenoids. Part 5. The preparation of 1-(3,3-dimethylallyl)-L-tryptophan and cyclo-L-aianyl-1-(3,3-dimethylallyl)-L-tryptophan and their non-incorporation into echinulin
“…190 12. Our results therefore support the current hypothesis for the mechanism of this reverse prenylation, as discussed by Williams et al14 Feeding experiments on echinulin,40 roquefortine,41, 42 paraherquamide A,15, 43 brevianamide A,15 and austamide15 also indicated that the reverse prenylation is more likely to take place by a simple S N 2′ alkylation.…”
A putative prenyltransferase gene-fgaPT1-has been identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus AF293. The gene was cloned and overexpressed in Escherichia coli, and the His6-fusion FgaPT1 was purified to near homogeneity and characterized biochemically. The enzyme was found to convert fumigaclavine A into fumigaclavine C by attaching a dimethylallyl moiety to C-2 of the indole nucleus in a "reverse" manner, that is, by connection of C-3 of the dimethylallyl moiety to an aromatic nucleus. FgaPT1 is a soluble, dimeric protein with a subunit size of 50 kDa. K m(app) values for fumigaclavine A and dimethylallyl diphosphate were determined to be 6 and 13 microM, respectively, while the turnover number was 0.8 s(-1). Metal ions such as Mg2+ and Ca2+ are not essential for the enzymatic activity. FgaPT1 showed relatively strict substrate specificity towards fumigaclavine A, with only dimethylallyl diphosphate being accepted as a donor under our conditions. FgaPT1 is the first reverse prenyltransferase from fungi to have been purified and characterized in homogenous form after heterologous overproduction. Surprisingly, it shows very low sequence similarity to the recently identified prenyltransferase LtxC from cyanobacteria, which also catalyzes the reverse prenylation of an indole nucleus.
“…190 12. Our results therefore support the current hypothesis for the mechanism of this reverse prenylation, as discussed by Williams et al14 Feeding experiments on echinulin,40 roquefortine,41, 42 paraherquamide A,15, 43 brevianamide A,15 and austamide15 also indicated that the reverse prenylation is more likely to take place by a simple S N 2′ alkylation.…”
A putative prenyltransferase gene-fgaPT1-has been identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus AF293. The gene was cloned and overexpressed in Escherichia coli, and the His6-fusion FgaPT1 was purified to near homogeneity and characterized biochemically. The enzyme was found to convert fumigaclavine A into fumigaclavine C by attaching a dimethylallyl moiety to C-2 of the indole nucleus in a "reverse" manner, that is, by connection of C-3 of the dimethylallyl moiety to an aromatic nucleus. FgaPT1 is a soluble, dimeric protein with a subunit size of 50 kDa. K m(app) values for fumigaclavine A and dimethylallyl diphosphate were determined to be 6 and 13 microM, respectively, while the turnover number was 0.8 s(-1). Metal ions such as Mg2+ and Ca2+ are not essential for the enzymatic activity. FgaPT1 showed relatively strict substrate specificity towards fumigaclavine A, with only dimethylallyl diphosphate being accepted as a donor under our conditions. FgaPT1 is the first reverse prenyltransferase from fungi to have been purified and characterized in homogenous form after heterologous overproduction. Surprisingly, it shows very low sequence similarity to the recently identified prenyltransferase LtxC from cyanobacteria, which also catalyzes the reverse prenylation of an indole nucleus.
“…Thus, aza-Claisen rearrangement of 27 would yield the reverse prenylated 3-indolenine 29 , which could subsequently suffer 1,2-migration of the dimethylvinyl carbon substituent followed by loss of the C-2 hydrogen atom of the indole nucleus to yield 28 . Separate experimental observations to probe this mode of reverse prenylation have cast some doubt on this type of mechanism for both roquefortine and the echinulins. ,− 2 …”
Section: Introductionmentioning
confidence: 99%
“…In the instance of the echinulins, Grundon et al concluded that the aza-Claisen type mechanism may not be operative in this system due to the lack of incorporation of the tritiated N -prenyl precursors 1-([1- 3 H]-3,3-dimethylallyl)- l -tryptophan and cyclo - l -alanyl-1-([1- 3 H]-3,3-dimethylallyl)- l -tryptophan (corresponding to 27 ) …”
The mode of attachment of dimethylallyl pyrophosphate (DMAPP) in the biosynthesis of the indole
alkaloids paraherquamide A, austamide, and brevianamide A has been studied. Feeding experiments on
Penicillium fellutanum, Penicillium brevicompactum, and Aspergillus
ustus using [13C2]-acetate showed isotopic
scrambling of the geminal methyl groups originating from C-2 of the indole ring precursors in paraherquamide
A, brevianamide A, and austamide biosynthesis. The labeling patterns suggest that the methyl groups of
dimethylallyl pyrophosphate become equivalent during the biosyntheses; a non-face-selective SN‘ mechanism
has been invoked to account for these observations.
“…Hamblin’s postdoctoral research was in biosynthesis carried out in two laboratories in the UK: first with Prof. Michael Grundon at the New University of Ulster; he studied the biosynthesis of the fungal metabolite echinulin [20] and then with Prof. Buchanan at Heriot Watt University; he studied the biosynthesis of the antiviral antibiotics, pyrazofurin [21] and showdomycin [22]. …”
Section: Dr Michael R Hamblin and His Team’s Contributionmentioning
The research interests of the Hamblin Laboratory are broadly centered on the use of different kinds of light to treat many different diseases. Photodynamic therapy (PDT) uses the combination of dyes with visible light to produce reactive oxygen species and kill bacteria, cancer cells and destroy unwanted tissue. Likewise, UV light is also good at killing especially pathogens. By contrast, red or near-infrared light can have the opposite effect, to act to preserve tissue from dying and can stimulate healing and regeneration. In all these applications, nanotechnology is having an ever-growing impact. In PDT, self-assembled nano-drug carriers (micelles, liposomes, etc.) play a great role in solubilizing the photosensitizers, metal nanoparticles can carry out plasmon resonance enhancement, and fullerenes can act as photosensitizers, themselves. In the realm of healing, single-walled carbon nanotubes can be electrofocused to produce nano-electonic biomedical devices, and nanomaterials will play a great role in restorative dentistry.
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