Dominik Pistorius

In Vivo Evidence for a Prodrug Activation Mechanism during Colibactin Maturation

Releasing the cytopath: We have identified an N-myristoyl-D-asparagine (1) as the free N-terminal prodrug scaffold in cytopathogenic Escherichia coli strains expressing the colibactin gene cluster. Colibactin is released in vivo upon cleavage of precolibactin. We provide for the first time in vivo evidence of the prodrug-like release mechanism of colibactin.

Pretubulysin, a Potent and Chemically Accessible Tubulysin Precursor from Angiococcus disciformis

Simplify, simplify, simplify! Pretubulysin (structure without the green substituents), a simplified tubulysin was prepared in the laboratory and also found in a natural myxobacterial source. This biosynthetic precursor of the tubulysins is not as active as tubulysins A and D but is still effective in picomolar concentrations against cancer cell lines.

Discovery of 23 Natural Tubulysins from Angiococcus disciformis An d48 and Cystobacter SBCb004

The tubulysins are a family of complex peptides with promising cytotoxic activity against multi-drug-resistant tumors. To date, ten tubulysins have been described from the myxobacterial strains Angiococcus disciformis An d48 and Archangium gephyra Ar 315. We report here a third producing strain, Cystobacter sp. SBCb004. Comparison of the tubulysin biosynthetic gene clusters in SBCb004 and An d48 reveals a conserved architecture, allowing the assignment of cluster boundaries. A SBCb004 strain containing a mutant in the putative cyclodeaminase gene tubZ accumulates pretubulysin A, the proposed first enzyme-free intermediate in the pathway, whose structure we confirm by NMR. We further show, using a combination of feeding studies and structure elucidation by NMR and high-resolution tandem mass spectrometry, that SBCb004 and An d48 together biosynthesize 22 additional tubulysin derivatives. These data reveal the inherently diversity-oriented nature of the tubulysin biosynthetic pathway.

Biosynthesis of 2‐Alkyl‐4(1H)‐Quinolones in Pseudomonas aeruginosa: Potential for Therapeutic Interference with Pathogenicity

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium capable of surviving in a broad range of natural environments and known to be involved in infectious diseases of various hosts. In humans, the opportunistic pathogen is one of the leading causes for nosocomial infections in immuno-compromised patients and is responsible for chronic lung infections in the majority of cystic fibrosis patients. The ability of P. aeruginosa to adapt to different environments and lifestyles is closely related to its ability to coordinate the survival strategy of a population by so-called quorum-sensing (QS) systems. QS is based on the production and release of small signaling molecules, called autoinducers, that increase in concentration as a function of cell density and activate corresponding transcriptional regulators after a threshold concentration has been reached. Three different QS systems are known from P. aeruginosa. The las and rhl systems use acyl-homoserine lactone (AHL) autoinducers and belong to the LuxI/LuxR-type systems that are widespread among Gram-negative bacteria. The third QS system is rather unique and restricted to particular Pseudomonas and Burkholderia strains. Therein 2-alkyl-4(1H)quinolones (AQ) autoinducers such as 2-heptyl-3-hydroxy4(1H)-quinolone (the Pseudomonas quinolone signal : PQS) and its direct precursor 2-heptyl-4(1H)-quinolone (HHQ) are used (see Scheme 1). The pqs system is involved in the regulation of P. aeruginosa virulence such as pyocyanin biosynthesis, biofilm formation and maturation, the production of exoproducts like elastase, alkaline proteases, rhamnolipids, and hydrogen cyanide, and the expression of efflux pumps. Further, PQS itself can down-regulate the host-innate immune response. The sum of these effects makes the pqs system a highly attractive target for drug development to interfere with P. aeruginosa pathogenicity and biofilm formation. The general validity of this approach is supported by the results of several infection models in which PQS-deficient mutants show a reduced pathogenicity compared to P. aeruginosa wild type. 10] A reduced pathogenicity was also observed in a mouse infection model when animals infected with the wild-type strain were treated with halogenated anthranilic acid derivatives that inhibit PQS biosynthesis. This treatment led to a significant increase in survival in comparison to the control group. To further explore this target it is necessary to understand the details of PQS formation in the pathogen. It is known that HHQ biosynthesis absolutely requires the genes pqsA–D, encoding an anthranilate:coenzyme A (CoA) ligase (pqsA) and three b-ketoacyl-acyl carrier protein synthase III (KAS III) homologues. An additional gene (pqsH), located apart from pqsA– D, is responsible for the hydroxylation of HHQ to form PQS. Feeding studies in vivo have demonstrated that HHQ most likely arises from “head-to-head” condensation of an anthraniloyl precursor and a b-keto fatty acid derivative (Scheme 1). However, the details of the enzymatic mechanism of this reaction and the nature of the b-keto fatty acid remained elusive. Furthermore, it has been shown in vitro that PqsA and PqsD catalyze the formation of 2,4-dihydroxyquinoline (DHQ), another secondary metabolite of P. aeruginosa. In DHQ biosynthesis PqsA activates anthranilic acid to anthraniloyl-CoA which is loaded to the active-site cysteine (C112) of PqsD, which itself catalyzes the decarboxylative Claisen condensation with malonyl-CoA. Based on the structural similarity between DHQ and HHQ, we reasoned that PqsD might be involved in a similar condensation reaction in HHQ biosynthesis. To prove this hypothesis, we heterologously expressed PqsD from strain PA14 in Escherichia coli and purified the enzyme for biochemical characterization in vitro. Anthraniloyl-CoA and three potential b-keto acid derivatives were chemically synthesized as substrates, including b-ketodecanoic acid (1), b-ketodecanoyl-CoA (2), and b-ketodecanoyl-N-acetylcysteamine thioester (3) as mimics of the hypothetical ACP-bound substrate (for details see the Supporting Information). The in vitro reaction contained recombinant PqsD, anthraniloyl-CoA, and one of the Scheme 1. Biosynthetic pathways to DHQ, HHQ, and PQS. The nature of the accepted b-ketodecanoyl moiety is unknown to date. Possible substrates are b-ketodecanoic acid (1), b-ketodecanoyl-CoA (2) and b-ketodecanoyl-ACP.

Discovery of the rhizopodin biosynthetic gene cluster in Stigmatella aurantiaca Sg a15 by genome mining.

The field of bacterial natural product research is currently undergoing a paradigm change concerning the discovery of natural products. Previously most efforts were based on isolation of the most abundant compound in an extract, or on tracking bioactivity. However, traditional activity‐guided approaches are limited by the available test panels and frequently lead to the rediscovery of already known compounds. The constantly increasing availability of bacterial genome sequences provides the potential for the discovery of a huge number of new natural compounds by in silico identification of biosynthetic gene clusters. Examination of the information on the biosynthetic machinery can further prevent rediscovery of known compounds, and can help identify so far unknown biosynthetic pathways of known compounds. By in silico screening of the genome of the myxobacterium Stigmatella aurantiaca Sg a15, a trans‐AT polyketide synthase/non‐ribosomal peptide synthetase (PKS/NRPS) gene cluster was identified that could not be correlated to any secondary metabolite known to be produced by this strain. Targeted gene inactivation and analysis of extracts from the resulting mutants by high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC‐HRMS), in combination with the use of statistical tools resulted in the identification of a compound that was absent in the mutants extracts. By matching with our in‐house database of myxobacterial secondary metabolites, this compound was identified as rhizopodin. A detailed analysis of the rhizopodin biosynthetic machinery is presented in this manuscript.

Human CYP4Z1 catalyzes the in-chain hydroxylation of lauric acid and myristic acid.

Abstract Overexpression of human CYP4Z1, a cytochrome P450 enzyme, has been correlated with poor prognosis in human cancer. However, its catalytic properties are not yet known. We expressed this P450 in Schizosaccharomyces pombe and demonstrate by whole-cell biotransformation assays CYP4Z1-dependent in-chain hydroxylation of lauric and myristic acid, which in both cases leads to the formation of four different monohydroxylated products at positions ω-2, ω-3, ω-4, and ω-5, respectively. The CYP4Z1-expressing fission yeast should be a new valuable tool for testing cancer drugs or for the development of new prodrug strategies.

AuaA, a Membrane-Bound Farnesyltransferase from Stigmatella aurantiaca, Catalyzes the Prenylation of 2-Methyl-4-hydroxyquinoline in the Biosynthesis of Aurachins

Aurachins are quinoline alkaloids isolated from the myxobacterium Stigmatella aurantiaca. They are substituted with an isoprenoid side chain and act as potent inhibitors in the electron transport chain. A biosynthetic gene cluster that contains at least five genes (auaA–auaE) has been identified for aurachin biosynthesis. In this study, auaA, the gene encoding a putative prenyltransferase of 326 amino acids, was cloned and overexpressed in Escherichia coli. Biochemical investigations showed that AuaA catalyzes the prenylation of 2‐methyl‐4‐hydroxyquinoline in the presence of farnesyl diphosphate (FPP), thereby resulting in the formation of aurachin D. The hydroxyl group at position C4 of the quinoline ring is essential for an acceptance by AuaA; this was concluded by testing 18 quinoline derivatives or analogues with AuaA and FPP. 1H NMR and HR‐EI‐MS analyses of six isolated enzyme products revealed the presence of a farnesyl moiety at position C3 of the quinoline ring. KM values of 43 and 270 μM were determined for FPP and 2‐methyl‐4‐hydroxyquinoline, respectively. Like other known membrane‐bound prenyltransferases, the reaction catalyzed by AuaA is dependent on the presence of metal ions such as Mg2+, Mn2+ and Co2+, although no typical (N/D)DXXD binding motif was found in the sequence.

Unprecedented anthranilate priming involving two enzymes of the acyl adenylating superfamily in aurachin biosynthesis.

Biosynthesis of many polyketide-derived secondary metabolites is initiated by incorporating starter units other than acetate. Thus, understanding their priming mechanism is of importance for metabolic engineering. Insight into the loading process of anthranilate into the biosynthetic pathway for the quinoline alkaloids aurachins has been provided by the sequencing of a partial biosynthetic gene cluster in the myxobacterium Stigmatella aurantiaca. The cluster encodes a predicted aryl:CoA ligase AuaE that was hypothesized to activate and transfer anthranilate to the acyl carrier protein AuaB. However, gene inactivation and in vitro experiments described here contradicted this model. Aided by the genome sequence of S. aurantiaca, we identified an additional aryl:CoA ligase homologue, AuaEII, encoded in a different gene operon, which is additionally required for anthranilate priming. We report the characterization of both enzymes and the elucidation of a novel non-acetate priming strategy in thio-templated biosynthetic machineries.

A semipinacol rearrangement directed by an enzymatic system featuring dual-function FAD-dependent monooxygenase.

Nature has invented ingenious ways to biosynthesize biologically active small molecules that have been applied ever since to benefit human life in various ways. During the underlying biosynthetic processes, highly elaborate chemical reactions are often catalyzed by enzymatic systems, thereby enabling transformations under physiological conditions that would require harsh conditions or are hardly possible without enzymatic catalysis. Consequently, understanding novel biochemical transformations is of importance to eventually apply the knowledge gained to generate molecules of interest. Aurachins are quinoline alkaloids isolated from the myxobacterium Stigmatella aurantiaca Sg a15; they have various biological activities, including antibacterial, antifungal, antiplasmodial, and mitochondrial respiration inhibition properties. The biosynthesis of aurachin derivatives includes several interesting features in which the most intriguing reaction is the conversion of aurachin C (1) to B (2). This step involves the migration of the prenyl group from position C3 to C4, probably via a pinacol type rearrangement (Scheme 1). Pinacol rearrangements are proposed to occur during the biosyntheses of various secondary metabolites, including aflatoxin B1, (+)-liphagal, (+)-asteltoxin, brevianamides, paraherquamide B, verscicolamide B, and notoamides. However, no such biosynthetic hypotheses has been biochemically proven, although the proposed pathways in turn inspired biomimetic approaches for natural product synthesis. Therefore, an enzymatic system for pinacol-type rearrangement remained to be discovered in the biosynthesis of secondary metabolites. Even in primary metabolism there are only two reported examples in course of the biosynthesis of branched chain amino acids and 1-deoxy-d-xylulose-5phosphate. The biosynthetic conversion of aurachin C (1) to B (2) was initially studied by feeding experiments carried out by Hçfle and Kunze. Importantly, they reported that the hydroxy group of aurachin B (2) at C3 is derived from molecular oxygen. Recent work by Pistorius et al. discovered two gene loci containing aurachin biosynthetic genes in addition to the core biosynthetic gene cluster. The authors speculated that two enzymes encoded by auaG and auaH are responsible for the migration of the farnesyl group from C3 to C4. However, the detailed biosynthetic conversion still remains to be solved, mainly because of the lack of detection of putative intermediates in vivo in auaG and auaH mutants of S. aurantiaca Sg a15. To uncover the enzymatic chemistry behind this intriguing rearrangement reaction, we here describe in vitro experiments using recombinant AuaG and AuaH proteins. AuaG belongs to the family of flavin-dependent monooxygenases and appears to be relatively similar to PgaE involved in angucycline biosynthesis according to results from BLAST search and Phyre2 analysis, respectively. The Scheme 1. Biosynthetic pathway of aurachin B (2) from aurachin C (1).

Flavonoid glycosides from the stem bark of Margaritaria discoidea demonstrate antibacterial and free radical scavenging activities

One new flavonoid glycoside, along with three known flavonoid glycosides were isolated from the stem bark of Margaritaria discoidea, which is traditionally used in the management of wounds and skin infections in Ghana. The new flavonoid glycoside was elucidated as hydroxygenkwanin‐8‐C‐[α‐rhamnopyranosyl‐(1 → 6)]‐β‐glucopyranoside (1) on the basis of spectroscopic analysis. The isolated compounds demonstrated free‐radical scavenging as well as some level of antibacterial activities. Microorganisms including Staphylococcus aureus are implicated in inhibiting or delaying wound healing. Therefore, any agent capable of reducing or eliminating the microbial load present in a wound as well as decreasing the levels of reactive oxygen species may facilitate the healing process. These findings therefore provide some support to the ethnopharmacological usage of the plant in the management of wounds. Copyright