Browsing by Subject "biosynthesis"

Now showing 1 - 3 of 3
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    Biochemical Interrogation of Polyketide Ketoreductase and Dehydratase Domain Stereoselectivity, Stereospecificity and Mechanism via Synthetic, Truncated Substrates
    (2016-04) Fiers, William
    Polyketide natural products are secondary metabolites produced in fungi, plants and bacteria. Since their discovery, these versatile small molecules have served as pharmaceuticals in many fields of medicine. From use as antibiotics to anti-cancer agents to immunosuppresants, polyketides remain staple components in the pharmacopeia. Nature biosynthesizes members of this natural product class through use of a complex network of enzymes known as polyketide synthases. There is an interest in studying enzymatic pathways that install chemical functional groups and unique three-dimensional form in hopes of rationally modifying them to create new drug molecules. This is an attractive prospect as enzyme catalysis can be predicted by genetic examination of the pathway. In theory, swapping, deleting or inserting a catalytic domain in the pathway would offer a means of controlled alteration of the natural product. While initial efforts have led to limited success, recently the focus has been shifted to understanding the mechanistic and structural details of these pathways with the aim of improving rational pathway diversification. Three aspects of polyketide synthesis: cryptic domains, distal stereochemistry and non- canonical domain architecture; remain relatively unexplored in the polyketide literature. Cryptic domains involve the configuration of polyketide intermediates obscured by later domain action. Stereochemistry distal to the site of manipulation on the polyketide may be a factor in pathway alteration, as the new catalytic site may not accept subtle changes made by prior enzymes. Modern genetics has revealed many pathways don’t follow the rules pertaining to the presence and order of catalytic domains. Bizarre exceptions to canonical domain architecture are difficult to reproduce and predict in modified pathways. Additionally, discovering how the product is ultimately produced may offer insight and new strategies for the coupling of polyketide synthase modules to create new products. We hypothesized that all three aspects of polyketide synthases (cryptic domains, distal stereochemistry and non-canonical domain architecture) could be studied through interrogation of individual catalytic domains with synthetic, diffusible substrates. In our studies we revealed several key elements of polyketide synthases that were previously unknown in the literature. We biochemically verified that cryptic stereochemistry and geometry can be accurately predicted based on genetic patterns in a polyketide synthase. A novel, mass spectrometry-based method of quantifying enzyme turnover in polyketide synthases was developed. This new technique allowed for the direct comparison of substrates with changes in distal stereochemistry in a dehydratase domain. Single inversions in configuration in substrates were found to result in a 14- to 45-fold loss in enzyme activity. Additionally, we were able to elucidate a unique mechanism for vinylogous dehydration in the curacin A pathway. This discovery explains why the polyketide synthase is missing domains and provides a clear exception to the notion of enzyme-product co-linearity. The combined work suggests that many potential pitfalls in the rational design of polyketide synthases can be anticipated and avoided through increased knowledge of pathway mechanisms and limitations.
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    Exploring Beta-Lactones as an Unexpected Link Between Hydrocarbon and Natural Product Biosynthesis in Bacteria
    (2017-05) Christenson, James
    Recent decades have seen a great interest in using biological systems as sources for the diverse chemicals used in modern society. The production of long-chain olefins (alkenes) from two fatty acids by diverse bacterial species has been well reported in literature and patented for the production of hydrocarbons. A four-gene cluster, oleABCD, encodes the enzymes responsible for this conversion; however, only two of the four enzymatic steps had been elucidated previously. Here, the third enzyme of the pathway, OleC, was characterized and found to convert beta-hydroxy acids to beta-lactones in an ATP-dependent reaction. beta-Lactone-containing natural products show great potential in clinical settings and are known for their potent inhibition of proteases and esterases. OleC is the first beta-lactone synthetase ever characterized, and sequence analysis suggests that the biosynthesis of other beta-lactone natural products is carried out by OleC homologues. The final enzymatic step of olefin biosynthesis was found to be the decarboxylation of the beta-lactone to an olefin by OleB. This enzymatic reaction was not known to exist in nature, making OleB the first known beta-lactone decarboxylase. OleB is frequently annotated as a haloalkane dehalogenase, but we find no dehalogenase activity, suggesting an incorrect annotation of OleB sequences. Mechanistic studies of OleB suggest a similar mechanism to haloalkane dehalogenases that relies on the same Asp-His-Asp catalytic triad and proceeds via an acyl-enzyme intermediate. Finally, the hydrophobic nature of the ole-pathway metabolites and the toxicity of the beta-lactone intermediate were hypothesized to require a direct transfer between the Ole enzymes. Accordingly, the four pathway enzymes, OleABCD, were recombinantly expressed in Escherichia coli, and OleBCD were found to form an enzyme complex. Immunoblots of OleC from the native Xanthomonas campestris on non-denaturing gels were found to migrate similarly to the recombinantly purified OleBCD complex and much slower than OleC by itself, suggesting formation of a complex in the native X. campestris host. Electron microscopy images showed distinct, circular assemblies of OleBCD between 24 and 40 nm in size, with an average size of 32 nm. The OleBCD complex functions to efficiently traffic the hydrophobic metabolites and sequester the toxic beta-lactone intermediate.
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    Protein structures elucidating the post-ribosomal biosynthesis of pyrroloquinoline quinone
    (2017-07) Evans III, Robert
    Not applicable