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Identification of the verruculogen prenyltransferase FtmPT3 by a combination of chemical, bioinformatic and biochemical approaches.
- Source :
-
Chembiochem : a European journal of chemical biology [Chembiochem] 2012 Nov 26; Vol. 13 (17), pp. 2583-92. Date of Electronic Publication: 2012 Oct 26. - Publication Year :
- 2012
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Abstract
- Previous studies showed that verruculogen is the end product of a biosynthetic gene cluster for fumitremorgin-type alkaloids in Aspergillus fumigatus and Neosartorya fischeri. In this study, we isolated fumitremorgin A from N. fischeri. This led to the identification of the responsible gene, ftmPT3, for O-prenylation of an aliphatic hydroxy group in verruculogen. This gene was found at a different location in the genome of N. fischeri than the identified cluster. The coding sequence of ftmPT3 was amplified by fusion PCR and overexpressed in Escherichia coli. The enzyme product of the soluble His(8)-FtmPT3 with verruculogen and dimethylallyl diphosphate (DMAPP) was identified unequivocally as fumitremorgin A by NMR and MS analyses. K(M) values of FtmPT3 were determined for verruculogen and DMAPP at 5.7 and 61.5 μM, respectively. Average turnover number (k(cat)) was calculated from kinetic parameters of verruculogen and DMAPP to be 0.069 s(-1). FtmPT3 also accepted biosynthetic precursors of fumitremorgin A, for example, fumitremorgin B and 12,13-dihydroxyfumitremorgin C, as substrates and catalyses their prenylation.<br /> (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Subjects :
- Chromosomes, Fungal genetics
Culture Techniques
Dimethylallyltranstransferase biosynthesis
Dimethylallyltranstransferase chemistry
Indenes metabolism
Kinetics
Multigene Family genetics
Neosartorya metabolism
Prenylation
Sequence Analysis
Substrate Specificity
Computational Biology
Dimethylallyltranstransferase genetics
Dimethylallyltranstransferase metabolism
Indoles metabolism
Neosartorya enzymology
Neosartorya genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1439-7633
- Volume :
- 13
- Issue :
- 17
- Database :
- MEDLINE
- Journal :
- Chembiochem : a European journal of chemical biology
- Publication Type :
- Academic Journal
- Accession number :
- 23109474
- Full Text :
- https://doi.org/10.1002/cbic.201200523