Studies on threonine synthase
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Threonine synthase (TS, EC 126.96.36.199), catalyses a β,γ-replacement reaction to convert (2S)-0-phosphohomoserine (32) into (2S,3S)-threonine (33). Although threonine as an essential amino acid is vital to all lifeforms, the enzyme is not expressed in mammals. This makes it an ideal target enzyme for herbicides, fungicides and bactericides, and several inhibitors of the enzyme have been produced with this in mind. In particular, the enzyme from Escherichia coli has previously been the subject of inhibition studies, and the reaction mechanism of this particular TS has also been partially elucidated. However, no product inhibition studies have been carried out on TS from E. coli. In order to carry out such studies, a [U-¹⁴C]-labelled version of the substrate (32b) was synthesised in this work, starting from [U-¹⁴C]-(2S)-aspartate (28b), via a route previously developed for producing the unlabelled substrate. Various analogues of the substrate have been synthesised and these were also to be tested with the enzyme, either as potential inhibitors, or to elucidate further the reaction mechanism and active-site structure of TS. The mutant E. coli strain K-12 Tir8, which had been used previously as a source of TS, appeared to have reverted to wild-type, no longer over-expressing the enzymes of the thr operon. Therefore, the thrC gene from E. coli, coding for TS, was cloned into a pET-expression system. In a host cell containing such a construct TS could amount to 50% of total cell protein. ThrC was amplified via PCR and inserted into a cloning vector, pGEM-T, and then subcloned into pET-3a, pET-3b and pET-16b. pET-16b constructs produce a His-tagged version of the recombinant protein. Sequencing of the recombinant gene in two pGEM-T constructs revealed one deletion and two mutations in the thrC sequence, which probably occurred during the PCR-amplification of the gene. These alterations were confirmed in sequences obtained for the pET-contructs. N,N-diisopropyldichlorophosphamidite (78), a precursor to a reagent used originally for the phosphorylation of ?-isopropyl-N-trifluoroacetyl-(2S)-homoserine (75), proved difficult to synthesise. Instead, dipentafluorophenyl phosphorochloridate (81) was used for the phosphorylation reaction, as it was easier to synthesise and gave a good yield of the phosphate ester. Deprotection of dipentafluorophenyl phosphates has, however, only been achieved previously on the solid-phase. The solid-phase synthesis of the substrate was therefore attempted using Wang, p-hydroxymethyl polystyrene and Merrifield resins. A new linker was attached to Merrifield, to produce the novel resin, polystyrene-4-oxymethyl-2-phenylethanol (97). Although selective opening of the N-trifIuoroacetyl-(25)-aspartic anhydride (73) was successfully accomplished to attach the α-carboxyl group to these resins, subsequent reduction of the β-acid has not been achieved. α-p-Benzyloxybenzylpolystyrene-N-trifluoroacetyl-(25)-aspartate (87) proved unstable towards reducing agents and bases. It is hoped that compounds attached to 97 will prove more stable towards reducing agents.
Thesis, PhD Doctor of Philosophy
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