Ergot alkaloid biosynthesis in Aspergillus fumigatus: overproduction and biochemical characterisation a 4-dimethylallyltryptop

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. INTRODUCTION Ergot alkaloids are a complex family of indole derivatives with diverse biological activities (1) and produced mainly by fungi of the genera Claviceps, Penicillium and Aspergillus (1,2). Ergot alkaloids can be divided into two classes of compounds according to their structural features: the clavines and D-lysergic acid amides (2,3). The clavines merely consist of the tetracyclic ergoline ring, e.g. agroclavine, fumigaclavines A and C (Figure 1) or its tricyclic seco derivatives, e.g. chanoclavine-I (Figure 1). D-lysergic acid-containing alkaloids, on the other hand, have substituents attached by amide bonds to the ergoline ring system. These substituents can be amino alcohols or tripeptide chains arranged in a unique bicyclic arrangement. The latter compounds are called ergopeptines, e.g. ergotamine (Figure 1), and are widely used as therapeutic agents (1). Ergot alkaloids of the clavine-type are also biologically active. For example, in vitro experiments have shown that fumigaclavine C has various pharmacological activities such as improvement of experimental colitis (4) and concanavalin A-induced liver injury in mice (5) as well as relaxation of isolated rat aortic rings (6). The clavine-type alkaloids fumigaclavines are http://www.jbc.org/cgi/doi/10.1074/jbc.M804979200 The latest version is at JBC Papers in Press. Published on August 4, 2008 as Manuscript M804979200 Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Jne 3, 2017 hp://w w w .jb.org/ D ow nladed from