Structural and chemical investigation of three biosynthetic enzymes
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Ribosomally synthesized and post-translationally modiﬁed peptides (RiPPs) are a burgeoning family of natural products with desirable bioactivities. The cyanobactin class of RiPPs have demonstrated anticancer, anti-microbial activities and more, and have therefore attracted research attentions. Cyanobactins are synthesised as a precursor peptide (PatE homologues) containing recognition sequences and core peptides (sequences that become the ﬁnal products), the latter of which undergo various modiﬁcations including heterocyclization, prenylation, oxidation and macrocyclization. The posttranslational modiﬁcation enzymes (PTMEs) utilised to modify the cyanobactin precursor have demonstrated magniﬁcent versatilities that are potentially exploitable for the creation of libraries of natural and unnatural compounds. The structure and function of each of these enzymes are reviewed in Chapter 1, along with the current application of the cyanobactin biosynthetic pathway. Chapter 2 describes my work on creating and characterising modiﬁed heterocyclases. The leader peptide was appended to heterocyclases that produce both thiazolines and (methyl)oxazolines. The modiﬁed catalysts are not only capable of utilising leaderless peptides, but also have higher yields than the native enzyme. The next chapter investigates the reaction order of heterocyclase enzymes. The leader peptide was found to be partially responsible for the reaction order. In Chapter 4 the same investigation was applied to the oxidase, which aromatises and stabilises the heterocycles. The oxidase reaction was determined to be independent of the leader. Chapter 5 delves into the phosphate chemistry of the heterocyclase, which uses ATP/Mg²⁺ via a kinase mechanism, but subsequently catalyses additional reactions that yield AMP and PPᵢ. Finally, Chapter 6 describes the structural characterisation of Psychrobacter arcticus ATP phosphoribosyltransferase (ATPPRT), which is the ﬁrst dedicated enzyme in histidine biosynthesis, and provides a key regulatory point for this pathway.
Thesis, PhD Doctor of Philosophy
Description of related resourcesMelo Czekster , C , Ge , Y & Naismith , J H 2016 , ' Mechanisms of cyanobactin biosynthesis ' , Current Opinion in Chemical Biology , vol. 35 , pp. 80-88 . https://doi.org/10.1016/j.cbpa.2016.08.029 (http://hdl.handle.net/10023/11669)
Stroek , R , Ge , Y , Talbot , P D , Glok , M K , Bernas , K E , Thomson , C M , Gould , E R , Alphey , M S , Liu , H , Florence , G J , Naismith , J H & da Silva , R G 2017 , ' Kinetics and structure of a cold-adapted hetero-octameric ATP phosphoribosyltransferase ' , Biochemistry , vol. 56 , no. 5 , pp. 793-803 . https://doi.org/10.1021/acs.biochem.6b01138 (http://hdl.handle.net/10023/12496)
Alphey , M S , Fisher , G , Ge , Y , Gould , E R , Guerreiro Machado , T F , Liu , H , Florence , G J , Naismith , J H & da Silva , R G 2018 , ' Catalytic and anticatalytic snapshots of a short-form ATP phosphoribosyltransferase ' , ACS Catalysis , vol. 8 , no. 6 , pp. 5601-5610 . https://doi.org/10.1021/acscatal.8b00867 (http://hdl.handle.net/10023/17684)
Gao , S , Ge , Y , Bent , A F , Schwarz-Linek , U & Naismith , J H 2018 , ' Oxidation of the cyanobactin precursor peptide is independent of the leader peptide and operates in a defined order ' , Biochemistry , vol. 57 , no. 41 , pp. 5996-6002 . https://doi.org/10.1021/acs.biochem.8b00835 ( http://hdl.handle.net/10023/18467)
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