Post-genome research on the biosynthesis of ergot ..
Ergot alkaloids, secondary metabolites produced by filamentous fungi, elicit a diverse array of pharmacological effects. The biosynthesis of this class of natural products has not been fully elucidated. Here we demonstrate that a homologue of Old Yellow Enzyme encoded in the ergot gene cluster catalyzes reduction of the α,β unsaturated alkene of chanoclavine-I aldehyde 3. This reduction, which yields dihydrochanoclavine aldehyde, facilitates an intramolecular reaction between a secondary amine and aldehyde to form the D ring of the ergot alkaloid structural framework.
Comparison of Ergot Alkaloid Biosynthesis Gene …
Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaDH has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaDH comprises 261 amino acids with a molecular mass of about 27.8 kDa and contains the conserved motifs of classical short-chain dehydrogenases/reductases (SDRs), but shares no worth mentioning sequence similarity with SDRs and other known proteins. The coding region of fgaDH consisting of two exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE60 and overexpressed in E. coli. The soluble tetrameric His6-FgaDH was purified to apparent homogeneity and characterized biochemically. It has been shown that FgaDH catalyzes the oxidation of chanoclavine-I in the presence of NAD+ resulting in the formation of chanoclavine-I aldehyde, which was unequivocally identified by NMR and MS analyzes. Therefore, FgaDH functions as a chanoclavine-I dehydrogenase and represents a new group of short-chain dehydrogenases. K M values for chanoclavine-I and NAD+ were determined at 0.27 and 1.1 mM, respectively. The turnover number was 0.38 s−1.
Genome sequencing provides new opportunities and challenges for identifying genes for the biosynthesis of secondary metabolites. A putative biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine type, was identified in the genome sequence of Aspergillus fumigatus by a bioinformatic approach. This cluster spans 22 kb of genomic DNA and comprises at least 11 open reading frames (ORFs). Seven of them are orthologous to genes from the biosynthetic gene cluster of ergot alkaloids in Claviceps purpurea. Experimental evidence of the identified cluster was provided by heterologous expression and biochemical characterization of two ORFs, FgaPT1 and FgaPT2, in the cluster of A. fumigatus, which show remarkable similarities to dimethylallyltryptophan synthase from C. purpurea and function as prenyltransferases. FgaPT2 converts L-tryptophan to dimethylallyltryptophan and thereby catalyzes the first step of ergot alkaloid biosynthesis, whilst FgaPT1 catalyzes the last step of the fumigaclavine C biosynthesis, i. e., the prenylation of fumigaclavine A at C-2 position of the indole nucleus. In addition to information obtained from the gene cluster of ergot alkaloids from C. purpurea, the identification of the biosynthetic gene cluster of fumigaclavine C in A. fumigatus opens an alternative way to study the biosynthesis of ergot alkaloids in fungi.