Ullrich Keller

Evidence for an ergot alkaloid gene cluster in Claviceps purpurea.

Abstract A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3′-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similiarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxido-reductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway.

Mechanism of alkaloid cyclopeptide synthesis in the ergot fungus Claviceps purpurea

BACKGROUND Previous analyses of the biosynthesis of the alkaloid cyclopeptides from the ergot fungus Claviceps purpurea were hampered by a lack of suitable systems for study in vitro, and this led to conflicting results concerning the mechanism of alkaloid cyclopeptide formation. Recently, D-lysergyl peptide synthetase (LPS) of the ergot fungus Claviceps purpurea, which assembles the non-cyclol precursors of the ergopeptines, has been partially purified and shown to consist of two polypeptide chains of 370 kDa (LPS 1) and 140 kDa (LPS 2); these contain all the sites necessary for the assembly of the D-lysergyl peptide backbone. The mechanism of D-lysergyl peptide synthesis remained unclear, however. RESULTS We have identified the obligatory peptidic intermediates in D-lysergyl peptide synthesis and the sequential order of their formation. The two LPS subunits catalyze the formation of D-lysergyl mono-, di-, and tripeptides as enzyme-thioester intermediates, the formation of which appears to be irreversible. Peptide synthesis starts when D-lysergic acid binds to the LPS 2 subunit, which most probably occurs after the previous round of synthesis has been completed by the release of the end product from the LPS enzyme. CONCLUSIONS We have shown that the mechanism of D-lysergyl peptide synthesis is an ordered process of successive acyl transfers on a multienzyme complex. This knowledge opens the way for enzymatic and genetic investigations into the formation of novel alkaloid cyclopeptides.

D-Lysergyl Peptide Synthetase from the Ergot Fungus Claviceps purpurea

The ergot fungus Claviceps purpurea produces the medically important ergopeptines, which consist of a cyclol-structured tripeptide and D-lysergic acid linked by an amide bond. An enzyme activity capable of non-ribosomal synthesis of D-lysergyl-L-alanyl-L-phenylalanyl-L-proline lactam, the non-cyclol precursor of the ergopeptine ergotamine, has been purified about 18-fold from the ergotamine-producing C. purpurea strain D1. Analysis of radioactively labeled enzyme-substrate complexes revealed a 370-kDa lysergyl peptide synthetase 1 (LPS 1) carrying the amino acid activation domains for alanine, phenylalanine, and proline. The activation of D-lysergic acid is catalyzed by a 140-kDa peptide synthetase (LPS 2) copurifying with LPS 1. LPS 1 and LPS 2 contain 4′-phosphopantetheine and bind their substrates covalently by thioester linkage. Kinetic analysis of the synthesis reaction revealed a Km of ∼1.4 µM for both D-lysergic acid and its structural homolog dihydrolysergic acid, which is one to two orders of magnitude lower than the Km values for the other amino acids involved. The Km values for the amino acids reflect their relative concentrations in the cellular pool of C. purpurea. This may indicate that in in vivo conditions D-lysergyl peptide formation is limited by the D-lysergic acid concentration in the cell. In vitro, the multienzyme preparation catalyzes the formation of several different D-lysergyl peptide lactams according to the amino acids supplied. Specific antiserum was used to detect LPS 1 in various C. purpurea strains. In C. purpurea wild type, the enzyme was expressed at all stages of cultivation and in different media, suggesting that it is produced constitutively.

Identification of the cytochrome P450 monooxygenase that bridges the clavine and ergoline alkaloid pathways.

Clavines and D‐lysergic acid‐derived alkaloid amides and alkaloid peptides are two different families of compounds that have the indole‐derived tetracyclic metergoline ring system in common. Previous work has shown that D‐lysergic acid is biosynthetically derived from clavine alkaloids. Recent cloning and analysis of the ergot alkaloid biosynthesis gene cluster from the D‐lysergic acid peptide (ergopeptines)‐producing Claviceps purpurea, has shown that it most probably contains all genes necessary for D‐lysergic acid synthesis as well as those that encode the assembly of D‐lysergic acid peptides, such as ergotamine. To address the role of the oxygenase genes of alkaloid‐gene clusters, the only cytochrome P450 monooxygenase gene of this cluster was inactivated by disruption. The resultant mutant accumulated agroclavine, elymoclavine, and chanoclavine in substantial amounts but not ergopeptines. Feeding the mutant with D‐lysergic acid restored ergopeptine synthesis; this suggests a block in the conversion of elymoclavine to D‐lysergic acid. The gene was designated cloA (for encoding a clavine oxidase, CLOA). Retransformation of the mutant with the intact cloA gene also restored ergopeptine synthesis. These data show that CLOA catalyses the conversion of clavines to D‐lysergic acid, it acts as a critical enzyme in the ergot alkaloid gene cluster, and bridges the biosynthesis of the two different families of alkaloids.

Functional Expression of the Ectoine Hydroxylase Gene (thpD) from Streptomyces chrysomallus in Halomonas elongata

ABSTRACT The formation of hydroxyectoine in the industrial ectoine producer Halomonas elongata was improved by the heterologous expression of the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus. The efficient conversion of ectoine to hydroxyectoine was achieved by the concerted regulation of thpD by the H. elongata ectA promoter.

Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides

BACKGROUND Many active peptides are synthesized by nonribosomal peptide synthetases (NRPSs), large multimodular enzymes. Each module incorporates one amino acid, and is composed of two domains: an activation domain that activates the substrate amino acid and a condensation domain for peptide-bond formation. Activation domains sometimes contain additional activities (e.g. N-methylation or epimerization). Novel peptides can be generated by swapping domains. Exchange of domains containing N-methylation activity has not been reported, however. RESULTS The actinomycin NRPS was used to investigate domain swapping. The first two amino acids of actinomycin are threonine and valine. We replaced the valine activation domain of module 2 with an N-methyl valine (MeVal) activation domain. The recombinant NRPS (AcmTmVe) catalyzes the formation of threonyl-valine. In the presence of S-adenosyl-methionine, valine was converted to MeVal but subsequent dipeptide formation was blocked. When acyl-threonine (the natural intermediate) was present at module 1, formation of acyl-threonine-MeVal occurred. The epimerization domain of AcmTmVe was impaired. CONCLUSIONS A simple activation domain can be replaced by one with N-methylation activity. The same condensation domain can catalyze peptide-bond formation between N-methyl and nonmethylated amino acids. Modification of the upstream amino acid (i.e. acylation of threonine), however, was required for condensation with MeVal. Steric hindrance reduces chemical reactivity of N-methyl amino acids - perfect substrate positioning may only be achieved with acylated threonine. Loss of the epimerase activity of AcmTmVe suggests N-methyltransferase and epimerase domains, not found together naturally, are incompatible.

Thiol Template Peptide Synthesis Systems in Bacteria and Fungi

Publisher Summary This chapter discusses the thiol template peptide synthesis systems in bacteria and fungi. Peptide synthetase systems are defined as the arrangement of various amino-acid activation domains in the form of a multi-enzyme or a multi-enzyme complex. This has been shown by comparisons of various enzyme systems with their corresponding genes. The order of the various activation domains is mirrored in the sequence of the peptide synthesized in prokaryotes. Eukaryotic peptide synthetases always consist of a single polypeptide chain encoded by an intronless gene. This single polypeptide chain harbours the various adenylate formation domains, thioester, and additional modules necessary for the synthesis of a given product. In synthetases from fungi, the peptide is assembled by aminoadipyl-cysteinyl-D-valine synthase (ACVS), which has been isolated from a representative number of fungi and bacteria. Based on biochemical investigations in the cases of the enzymes from A. nidulans and S. clavuligerus and also considering the sequences of a number of ACVS genes, it is clear that this enzyme is composed of three peptide synthetase domains lying on one polypeptide chain of 420 kDa. Much progress in the enzymology of prokaryotic peptide synthetases has been achieved in the field of the acyl peptide lactone synthetases. Acyl peptide lactones consist of peptide lactone rings to which are attached aromatic or aliphatic side groups in an amide-like fashion.

The Actinomycin Biosynthetic Gene Cluster of Streptomyces chrysomallus: a Genetic Hall of Mirrors for Synthesis of a Molecule with Mirror Symmetry

A gene cluster was identified which contains genes involved in the biosynthesis of actinomycin encompassing 50 kb of contiguous DNA on the chromosome of Streptomyces chrysomallus. It contains 28 genes with biosynthetic functions and is bordered on both sides by IS elements. Unprecedentedly, the cluster consists of two large inverted repeats of 11 and 13 genes, respectively, with four nonribosomal peptide synthetase genes in the middle. Nine genes in each repeat have counterparts in the other, in the same arrangement but in the opposite orientation, suggesting an inverse duplication of one of the arms during the evolution of the gene cluster. All of the genes appear to be organized into operons, each corresponding to a functional section of actinomycin biosynthesis, such as peptide assembly, regulation, resistance, and biosynthesis of the precursor of the actinomycin chromophore 4-methyl-3-hydroxyanthranilic acid (4-MHA). For 4-MHA synthesis, functional analysis revealed genes that encode pathway-specific isoforms of tryptophan dioxygenase, kynurenine formamidase, and hydroxykynureninase, which are distinct from the corresponding enzyme activities of cellular tryptophan catabolism in their regulation and in part in their substrate specificity. Phylogenetic analysis indicates that the pathway-specific tryptophan metabolism in Streptomyces most probably evolved divergently from the normal pathway of tryptophan catabolism to provide an extra or independent supply of building blocks for the synthesis of tryptophan-derived secondary metabolites.

Biosynthesis of Ergotamine in Protoplasts of Claviceps purpurea

Protoplasts of Claviceps purpurea (ATCC 20102) were prepared in 0.8 m-sucrose containing 10 mm-CaCl2 and 10 mm-MgCl2. Protoplasts could revert to the filamentous state but not after treatment with water. Most of the protoplasts (about 80%) were highly vacuolated and these were separated from the non-vacuolated protoplasts and cell debris on the basis of their low density. Only the vacuolated protoplasts were able to synthesize ergotamine and ergocryptine de novo. Protoplasts were about 50% less active than the control mycelium. The control mycelium was more active in the uptake of labelled precursors than both protoplasts and freshly harvested mycelium. In the amino acid pool of protoplasts, alanine was present in a concentration which exceeded that of proline by a factor of six and that of phenylalanine by a factor of 100. This finding is consistent with the incorporation ratios of these amino acids into ergotamine when isotope dilution of the added radiolabel is considered. A significant stimulation of incorporation of constituent amino acids into ergotamine and ergocryptine occurred when d-lysergic acid was added to protoplasts and mycelium.

Cyclolization of D-Lysergic Acid Alkaloid Peptides

The tripeptide chains of the ergopeptines, a class of pharmacologically important D-lysergic acid alkaloid peptides, are arranged in a unique bicyclic cyclol based on an amino-terminal α-hydroxyamino acid and a terminal orthostructure. D-lysergyl-tripeptides are assembled by the nonribosomal peptide synthetases LPS1 and LPS2 of the ergot fungus Claviceps purpurea and released as N-(D-lysergyl-aminoacyl)-lactams. We show total enzymatic synthesis of ergopeptines catalyzed by a Fe²⁺/2-ketoglutarate-dependent dioxygenase (EasH) in conjunction with LPS1/LPS2. Analysis of the reaction indicated that EasH introduces a hydroxyl group into N-(D-lysergyl-aminoacyl)-lactam at α-C of the aminoacyl residue followed by spontaneous condensation with the terminal lactam carbonyl group. Sequence analysis revealed that EasH belongs to the wide and diverse family of the phytanoyl coenzyme A hydroxylases. We provide a high-resolution crystal structure of EasH that is most similar to that of phytanoyl coenzyme A hydroxylase, PhyH, from human.