Title

Identification of the Verruculogen Prenyltransferase FtmPT3 by a Combination of Chemical, Bioinformatic and Biochemical Approaches

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.

Authors

Kathrin Mundt, Beate Wollinsky, Han-Li Ruan, Tianjiao Zhu, Shu-Ming Li

Journal

ChemBioChem

Year, Volume, Page

2012, 13, 2583

Reactions

3



Tag Element Regiochemistry Product Substrate Cofactor Enzyme
PTDBREC00031 N Regular FtmPT3
PTDBREC00032 O Regular FtmPT3
PTDBREC00033 O Regular FtmPT3