Unusual Building Blocks and Domain Organization of Non-Ribosomal Peptide Synthetases

The diverse class of non-ribosomal peptides consists of manifold pharmacologically important natural products. They are clinically used in antibiotic, antiviral and antitumor therapy, furthermore some are known immunosuppresants. The biological activity is based on their structural diversity, as they contain various non-proteinogenic building blocks and amino acids of which many are beta-modified. It was shown that the latter are important for biological activity, but little is known about their biosynthetic origin. In particular, these building blocks are key determinants of the class of acidic lipopeptide antibiotics and kutznerides, which are in the focus of this thesis. To determine the mechanism underlying the biosynthetic origin of the synthetically challenging beta-hydroxylated asparagine (hAsn) moieties, found in the acidic lipopeptides CDA and A54145, the corresponding recombinant non-heme iron (II)/alpha-ketoglutarate dependent hydroxylases AsnO and LptL have been examined in vitro. Direct hydroxylation of the free amino acid was observed in both cases, clearly indicating a precursor synthesis pathway. The crystal structure of one of the two hydroxylases (AsnO) was determined at high resolution and revealed a substrate induced fit mechanism of the enzyme. Upon addition of asparagine, a lid-like region seals the active site and shields it from sterically demanding substrates, which explains the observed specificity for free asparagine. Furthermore, the AsnO structure could be seen as an archetype enzyme for non-heme iron hydroxylases acting on free amino acids. It was possible to predict amino acid binding residues for homologous enzymes by 3D modeling. In order to fully understand the mechanisms of beta-hydroxylated building blocks synthesis, the hydroxylases KtzO and KtzP, predicted to be responsible for the generation of the two 3-hydroxyglutamic acid isomers found in the mixture of antifungal and antimicrobial kutznerides, were produced recombinantly and analyzed in vitro. Notably, they were found to work in trans to the assembly line on PCP-tethered glutamic acid rather than on the free amino acid. Unexpectedly, as the two isomers are found in approximately equal amounts in mature kutznerides, KtzO was shown to stereospecifically generate threo-hydroxyglutamate, while KtzP catalyzed the formation of the erythro isomer by co-elution HPLC experiments with synthetic dabsylated standards. A powerful method that employs non-hydrolyzable coenzyme A analogs was developed, which allowed the determination of the kinetic parameters of enzymes working on PCP-bound substrates for the first time. Furthermore, a hitherto unknown mechanism of NRPS assembly line restoration was observed. The corresponding adenylation (A) domain for glutamic acid activation in the kutzneride NRPS was found to be corrupted. Herein, it is shown that this lack of a functional A domain is compensated in trans by a stand-alone A domain. These findings elucidated the mechanism for the in trans compensation and the stereospecific hydroxyglutamate generation in detail and may guide the usage of in trans hydroxylation/compensation enzymes in biocombinatorial engineering approaches. In the third part of this work, the acquired knowledge about the mechanisms underlying enzymatic beta-hydroxylation of amino acids was exploited for the synthesis of the pharmaceutically relevant beta-hydroxyaspartate. Primarily, this was facilitated by the structure elucidation of AsnO in which the substrate binding residues were identified. By site directed mutagenesis, an AsnO variant was generated, which notably did not hydroxylate the original substrate asparagine, instead it was found to stereospecifically catalyze the formation of L-threo-hydroxyaspartic acid, even in commercially interesting amounts. Therefore, the AsnO variant is an excellent example for the application of basic research in order to generate pharmacologically relevant non-proteinogenic amino acids

Iron is a centrally bound cofactor of specifier proteins involved in glucosinolate breakdown

Glucosinolates, a group of sulfur-rich thioglucosides found in plants of the order Brassicales, have attracted a lot of interest as chemical defenses of plants and health promoting substances in human diet. They are accumulated separately from their hydrolyzing enzymes, myrosinases, within the intact plant, but undergo myrosinase-catalyzed hydrolysis upon tissue disruption. This results in various biologically active products, e.g. isothiocyanates, simple nitriles, epithionitriles, and organic thiocyanates. While formation of isothiocyanates proceeds by a spontaneous rearrangement of the glucosinolate aglucone, aglucone conversion to the other products involves specifier proteins under physiological conditions. Specifier proteins appear to act with high specificity, but their exact roles and the structural bases of their specificity are presently unknown. Previous research identified the motif EXXXDXXXH as potential iron binding site required for activity, but crystal structures of recombinant specifier proteins lacked the iron cofactor. Here, we provide experimental evidence for the presence of iron (most likely Fe2+) in purified recombinant thiocyanate-forming protein from Thlaspi arvense (TaTFP) using a Ferene S-based photometric assay as well as Inductively Coupled Plasma-Mass Spectrometry. Iron binding and activity depend on E266, D270, and H274 suggesting a direct interaction of Fe2+ with these residues. Furthermore, we demonstrate presence of iron in epithiospecifier protein and nitrile-specifier protein 3 from Arabidopsis thaliana (AtESP and AtNSP3). We also present a homology model of AtNSP3. In agreement with this model, iron binding and activity of AtNSP3 depend on E386, D390, and H394. The homology model further suggests that the active site of AtNSP3 imposes fewer restrictions to the glucosinolate aglucone conformation than that of TaTFP and AtESP due to its larger size. This may explain why AtNSP3 does not support epithionitrile or thiocyanate formation, which likely requires exact positioning of the aglucone thiolate relative to the side chain

Production of benzylglucosinolate in genetically engineered carrot suspension cultures.

Glucosinolates, a group of sulfur-containing specialized metabolites of the Brassicales, have attracted a lot of interest in nutrition, medicine and agriculture due to their positive health effects and their involvement in plant defense. Their biological activities and the extensive knowledge of their biosynthesis have inspired research into development of crops with enhanced glucosinolate contents as well as their biotechnological production in homologous and heterologous systems. Here, we provide proof-of-concept for transgenic suspension cultures of carrot (Daucus carota, Apiacae) as a scalable production platform for plant specialized metabolites using benzylglucosinolate as a model. Two T-DNAs carrying in total six genes of the benzylglucosinolate biosynthesis pathway from Arabidopsis thaliana as well as NPTII and BAR as selectable markers were transferred to carrot cells by Agrobacterium tumefaciens-mediated transformation. Putative transformants selected based on their kanamycin and BASTA resistances were subjected to HPLC-MS analysis. Of 79 putative transformants, 17 produced benzylglucosinolate. T-DNA-integration was confirmed for the five best producers. Callus from these transformants was used to establish suspension cultures for quantitative analysis. When grown in 60-ml-cultures, the best transformants produced roughly 2.5 nmol (g fw)-1 benzylglucosinolate, together with up to 10 nmol (g fw)-1 desulfobenzylglucosinolate. Only one transformant produced more benzylglucosinolate than desulfobenzylglucosinolate. The concentration of sulfate in the medium was not a major limiting factor. High production seemed to be associated with poor growth and vice versa. Therefore, future research should try to optimize medium and cultivation process and to separate growth and production phase by using an inducible promoter

Unusual Building Blocks and Domain Organization of Non-Ribosomal Peptide Synthetases

The diverse class of non-ribosomal peptides consists of manifold pharmacologically important natural products. They are clinically used in antibiotic, antiviral and antitumor therapy, furthermore some are known immunosuppresants. The biological activity is based on their structural diversity, as they contain various non-proteinogenic building blocks and amino acids of which many are beta-modified. It was shown that the latter are important for biological activity, but little is known about their biosynthetic origin. In particular, these building blocks are key determinants of the class of acidic lipopeptide antibiotics and kutznerides, which are in the focus of this thesis. To determine the mechanism underlying the biosynthetic origin of the synthetically challenging beta-hydroxylated asparagine (hAsn) moieties, found in the acidic lipopeptides CDA and A54145, the corresponding recombinant non-heme iron (II)/alpha-ketoglutarate dependent hydroxylases AsnO and LptL have been examined in vitro. Direct hydroxylation of the free amino acid was observed in both cases, clearly indicating a precursor synthesis pathway. The crystal structure of one of the two hydroxylases (AsnO) was determined at high resolution and revealed a substrate induced fit mechanism of the enzyme. Upon addition of asparagine, a lid-like region seals the active site and shields it from sterically demanding substrates, which explains the observed specificity for free asparagine. Furthermore, the AsnO structure could be seen as an archetype enzyme for non-heme iron hydroxylases acting on free amino acids. It was possible to predict amino acid binding residues for homologous enzymes by 3D modeling. In order to fully understand the mechanisms of beta-hydroxylated building blocks synthesis, the hydroxylases KtzO and KtzP, predicted to be responsible for the generation of the two 3-hydroxyglutamic acid isomers found in the mixture of antifungal and antimicrobial kutznerides, were produced recombinantly and analyzed in vitro. Notably, they were found to work in trans to the assembly line on PCP-tethered glutamic acid rather than on the free amino acid. Unexpectedly, as the two isomers are found in approximately equal amounts in mature kutznerides, KtzO was shown to stereospecifically generate threo-hydroxyglutamate, while KtzP catalyzed the formation of the erythro isomer by co-elution HPLC experiments with synthetic dabsylated standards. A powerful method that employs non-hydrolyzable coenzyme A analogs was developed, which allowed the determination of the kinetic parameters of enzymes working on PCP-bound substrates for the first time. Furthermore, a hitherto unknown mechanism of NRPS assembly line restoration was observed. The corresponding adenylation (A) domain for glutamic acid activation in the kutzneride NRPS was found to be corrupted. Herein, it is shown that this lack of a functional A domain is compensated in trans by a stand-alone A domain. These findings elucidated the mechanism for the in trans compensation and the stereospecific hydroxyglutamate generation in detail and may guide the usage of in trans hydroxylation/compensation enzymes in biocombinatorial engineering approaches. In the third part of this work, the acquired knowledge about the mechanisms underlying enzymatic beta-hydroxylation of amino acids was exploited for the synthesis of the pharmaceutically relevant beta-hydroxyaspartate. Primarily, this was facilitated by the structure elucidation of AsnO in which the substrate binding residues were identified. By site directed mutagenesis, an AsnO variant was generated, which notably did not hydroxylate the original substrate asparagine, instead it was found to stereospecifically catalyze the formation of L-threo-hydroxyaspartic acid, even in commercially interesting amounts. Therefore, the AsnO variant is an excellent example for the application of basic research in order to generate pharmacologically relevant non-proteinogenic amino acids

The Impact of Nitrile-Specifier Proteins on Indolic Carbinol and Nitrile Formation in Homogenates of Arabidopsis thaliana

Glucosinolates, specialized metabolites of the Brassicales including Brassica crops and Arabidopsis thaliana, have attracted considerable interest as chemical defenses and health-promoting compounds. Their biological activities are mostly due to breakdown products formed upon mixing with co-occurring myrosinases and specifier proteins, which can result in multiple products with differing properties, even from a single glucosinolate. Whereas product profiles of aliphatic glucosinolates have frequently been reported, indole glucosinolate breakdown may result in complex mixtures, the analysis of which challenging. The aim of this study was to assess the breakdown of indole glucosinolates in A. thaliana root and rosette homogenates and to test the impact of nitrile-specifier proteins (NSPs) on product profiles. To develop a GC-MS-method for quantification of carbinols and nitriles derived from three prominent indole glucosinolates, we synthesized standards, established derivatization conditions, determined relative response factors and evaluated applicability of the method to plant homogenates. We show that carbinols are more dominant among the detected products in rosette than in root homogenates of wild-type and NSP1- or NSP3-deficient mutants. NSP1 is solely responsible for nitrile formation in rosette homogenates and is the major NSP for indolic nitrile formation in root homogenates, with no contribution from NSP3. These results will contribute to the understanding of the roles of NSPs in plants