Ergot alkaloid biosynthesis in Aspergillus fumigatus: overproduction and biochemical characterisation of a 4-dimethylallyltryptophan N-methyltransferase
Author:
Rigbers O, Li SM
Date: 4 September 2008
Abstract:
A putative gene fgaMT was identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus. The coding region of fgaMT was amplified by PCR from a cDNA library, cloned into pQE60 and overexpressed in E. coli. FgaMT comprises 339 amino acids with a molecular weight of about 38.1 kDa. The soluble dimeric His6-FgaMT was purified to near homogeneity and characterised biochemically. FgaMT was found to catalyse the N-methylation of 4-dimethylallyltryptophan in the presence of S-adenosylmethionine resulting in the formation of 4-dimethylallyl-L-abrine, which was identified by NMR and MS analysis. Therefore, FgaMT represents the second pathway-specific enzyme in the biosynthesis of ergot alkaloids. The enzyme did not require metal ions for its enzymatic reaction and showed a relatively high specificity towards the prenyl moiety at position C-4 of the indole ring. 4-dimethylallyltryptophan derivatives with modification at the indole ring were also accepted by FgaMT as substrates. Km values for 4-dimethylallyltryptophan and S-adenosylmethionine were determined at 0.12 and 2.4 mM, respectively. The turnover number was 2.0 s-1.
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