Paderborn, Universiẗat, Diss., 2005.

No description available.

No description available.

No description available.

Polyketides are a class of small molecules synthesized by a broad spectrum of bacteria, plants, and fungi, and many exhibit powerful bioactive properties. The number of clinically-relevant compounds adapted from polyketide scaffolds is growing, eliciting attempts from synthetic organic chemists to construct polyketide-related compounds in the laboratory from simple chemical building blocks. Unfortunately, the current efficiency by which a skilled artisan can synthesize even small quantities of a polyketide is severely limited by the functional and stereochemical complexity of these compounds. Conceptually, it would be much simpler to genetically reprogram the enzymes responsible for polyketide biosynthesis to produce designer molecules; however, the massive size of polyketide synthase enzymes has hindered efforts towards understanding critical features of their structures and mechanisms. Only very recently has structural information become available for enzymes involved in polyketide biosynthesis, providing an initial glimpse into the inner workings of these subcellular pharmaceutical factories. It will not be possible for mankind to fully realize the potential of engineered polyketide synthases without understanding how their architectures govern the molecules they have evolved to produce. In this work, the structure and mechanism of several enzymes involved in polyketide biosynthesis is investigated. An unprecedented architecture for the ketoreductase-enoylreductase didomain from the second module of the spinosyn polyketide synthase reveals structural divergence from the related mammalian fatty acid synthase, and reconstituted in vitro activity of the enoylreductase domain indicates the isolated enzyme retains activity apart from its parent polyketide synthase module. The dehydratase domain isolated from the tenth module of the rifamycin polyketide synthase, previously hypothesized to only form double bonds with (Z) geometry, was found to have altered stereoselectivity dependent on the carrier handle bound to the substrate. The enoyl-isomerase domain, isolated from the fourteenth module of the bacillaene polyketide synthase, utilizes a catalytic mechanism that relies only on a single active site histidine. A series of ketosynthase domains from trans-acyltransferase polyketide synthases reveal how polyketides bind covalently to the active site of the ketosynthase, and how the flanking subdomain of the ketosynthase is used as an anchor point for the assembly of the polyketide synthase megacomplex.

Göttingen, Universiẗat, Diss., 2000.

No description available.

The goals of this work are to advance the understanding of the mechanisms of thioesterase loading-substrate and release-nucleophile selectivity and to develop an analytical methodology and modelling theory as tools for quantitative measures of thioesterase activity. The first aspect of this work is a bioinformatic examination of examples of thioesterases that catalyze different forms of release at different rates of reaction dependent on the type of thioester substrate presented to it. In addition, the type I thioesterase domain from the Type I modular polyketide synthase pathway responsible for the production of bacillaene was cloned, expressed, and purified for kinetic characterization as a model trans-AT type thioesterase. N-Acetylcysteamine and acyl carrier protein-bound acyl substrates were synthesized as substrates for hydrolysis catalysed by the bacillaene thioesterase. The rate of reaction was indirectly observed with a widely-adopted visual spectroscopy assay facilitated by the thiol indicator 5,5'-dithio-bis-[2-nitrobenzoic acid]. Variants of the bacillaene thioesterase coded from two different bacterial isolates with 57 % amino acid identity (73 % similarity) both exhibited rapid declining activity under the conditions used for Ellman’s, indicative of inhibition. This matches rare literature examples of other thioesterase domains under the same spectroscopic assay conditions. We demonstrate that initial rate of reaction data is insufficient to describe substrate selectivity in these systems and develop a time-dependent model capable of accounting for irreversible substrate inhibition during catalysis. This model was sufficient to reproduce the thioesterase activity observed between the bacillaene thioesterase and methyl valeric-SNAC but is insufficient to model the responses observed during the hydrolysis of trans-2-methyl-2-pentenoyl-SNAC or cis-3-methyl-2-pentenoyl-SNAC. Finally, we detailed synthetic and assay conditions for the production, cleavage, and detection of the ACP-tethered substrate equivalents. This contributes towards the development of complex thioesterase assays that better estimate type I bacterial thioesterase activity during polyketide synthesis.

6-Methylsalicylic acid (6-MSA) is one of the oldest known polyketides. It is synthesised in vivo by the polyketide synthase 6-methylsalicylic acid synthase (6-MSAS), a multifunctional enzyme which uses its active sites iteratively. The stereochemistry of the hydroxyl produced from the single ketoreduction, as well as the order of dehydration, cyclisation and aromatisation steps, remain cryptic, despite extensive study. Holo 6-MSAS was heterologously expressed in E. coli and purified in two steps. A non-hydrolysable carba(dethia)malonyl-N-acetylcysteamine analogue was synthesised and used to off-load enzyme-bound intermediates from 6-MSAS. In assays with acetyl-CoA and acetoacetyl-CoA alone, diketide and triketide intermediates were off-loaded and detected by HPLC-HR-ESI-MS. In the presence of NADPH, the off-loaded triketide was reduced by the ketoreductase domain of 6-MSAS. A potential dehydrated intermediate was also observed. The dehydratase domain of 6-MSAS has recently been reassigned as a thioester hydrolase. To test this theory, the catalytic histidine residue in 6-MSAS was mutated to an alanine and the abolition of production of 6-MSA in vivo was observed. Mutated 6-MSAS was still able to produce the shunt product triacetic acid lactone. Incubation of mutated 6-MSAS with acetyl-CoA, malonyl-CoA, NADPH and carba(dethia)malonyl-N-acetylcysteamine saw only the off-loading of diketide and triketide analogues. To investigate the stereochemistry of ketoreduction in 6-MSA biosynthesis, steps were made to synthesise the resolved diastereomeric reduced-triketide CoAs which would be the substrates for the ketoreductase domain. Attempts to phosphopantetheinylate apo 6-MSAS in vitro with three different phosphopantetheinyltransferases were unsuccessful. Limited proteolysis of both holo and apo 6-MSAS found that the apo synthase rapidly lost a C-terminal fragment while holo 6-MSAS was much more stable under the same conditions. Attempts were made to express the acyl carrier protein domain from 6-MSAS to overcome these problems. These experiments represent the first use of the non-hydrolysable analogue methodology in a Type I iterative polyketide synthase and provide a framework for future experiments investigating intermediates in the biosynthesis of 6-MSA.

Macrocyclic natural products are conformationally restricted molecules that often have improved ability to bind with high affinity and selectivity on a target. Within macrocycle chemistry, macrolactone formation is a particularly challenging transformation and has spurred the development of highly diverse synthetic strategies. A key strategy that is missing is a chemoenzymatic approach to this challenge, and the logical place to look for such a catalyst is the thioesterases (TEs) from the biosynthetic pathways that generate these molecules in Nature. These TEs are α/β-hydrolases containing an active site serine or cysteine and a conserved histidine/aspartate catalytic diad. The research presented here describes the development of two related TE domains from resorcylic acid lactone polyketide synthases found in various fungi. Unlike their bacterial counterparts these macrocyclization catalysts have proven to be stereotolerant with regard to the secondary alcohols involved in macrocyclization. Further work has demonstrated that they are also amenable to generating 12- to 18-member macrolactones. These TE domains can also catalyze macrolactam and cyclic depsipeptide formation, setting the stage for these enzymes to serve as a platform for catalyst development. The development of 2,3-diaminopropionate (DAP) incorporation in place of the active site Ser to trap acyl-enzyme intermediates was used to structurally characterize the formation of a macrocyclic trimer. This technique will be broadly applicable to characterizing other TEs. Overall the research presented here lays the foundation for the long term development of TEs as macrocyclization biocatalysts.