A Study of Two Key Enzymes in the Diaminopimelate Pathway to L-Lysine

Tudor, David Wilson (1992) A Study of Two Key Enzymes in the Diaminopimelate Pathway to L-Lysine. PhD thesis, University of Glasgow.

Abstract

There are two distinct biosynthetic pathways to the essential amino acid L-lysine (A). The diaminopimelate pathway to L-lysine (A) occurs in higher plants and bacteria whereas the alpha-aminoadipate pathway to L-lysine (A) operates in fungi and yeasts. This thesis is concerned with the first two steps in the diaminopimelate pathway to L-lysine (A), catalysed by dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR). In particular, the synthesis of L-aspartic acid-beta-semialdehyde (L-ASA) (C), a substrate of the first enzyme and the mechanism of formation of L-2,3-dihydrodipicolinate (L-2,3-DHDPA) (B), the product of the reaction catalysed by DHDPS, was studied. In addition the synthesis of compounds which might be inhibitors of DHDPS was studied. L-Aspartic acid-beta-semialdehyde (C) is an important intermediate in the biosynthesis of L-lysine (A), L-threonine and L-methionine. The trifluoroacetate salts of the L- and D-isomers of aspartic acid-beta-semialdehyde were prepared from L- and D-allylglycine respectively. Our Biochemistry co-workers have isolated and purified DHDPS and have set up an assay system for this enzyme. Using this assay system we confirmed that L-ASA (C) is a substrate for DHDPS whereas the D-isomer is not. Having pure L-ASA (C) allowed us to carry out precise NMR spectroscopic experiments and biochemical experiments to investigate the mechanism of DHDPS. We confirmed that the product from the enzymic reaction is dipicolinic acid (D). Manipulation of the synthetic route to L-ASA (C) allowed us to prepare a range of aspartic acid-?-semialdehyde analogues and derivatives. Most of these compounds were tested for potential inhibitor or substrate activity with DHDPS. In particular the trifluoroacetate salt of DL-aspartic acid-?-semialdehyde methyl ester hydrate (E) showed no substrate activity however, 14% inhibition was observed with this compound at 0.5 mM. The other substrate of DHDPS in the diaminopimelate pathway to L-lysine (A) is pyruvate (F). A range of pyruvate (F) and bromopyruvate derivatives were synthesised and tested as substrates or inhibitors of DHDPS. Methyl pyruvate has shown substrate activity with DHDPS. In general, the bromopyruvate derivatives were better inhibitors than the pyruvate derivatives.A range of sulphur analogues of L-2,3-DHDPA (B) and L-2,3,4,5-tetrahydrodipicolinic acid (L-2,3,4,5-THDPA) (G) were synthesised and tested for inhibitor activity with DHDPS. The 3,4-dihydro-1,4-thiazines, in particular the diethyl ester (H) and its iV-methyl analogue (I), showed good inhibition at 0.1 mM with DHDPS. The 3,4-dihydro-2,2-dimethyl-1,4-thiazine diester (J) showed the best inhibition with 20% at 0.1 mM. The 3,4-dihydro-1,4-thiazines were prepared by reacting L-cysteine derivatives with ethyl bromopyruvate in dichloromethane in the presence of triethylamine. The 1,3-thiazoles that were prepared showed very good inhibition with DHDPS. The diester (K) showed 21% inhibition at 0.1mM with DHDPS. This compound was prepared by treatment of the mercapto-amide (L) with phosphorus pentasulphide in pyridine. The mercapto-amide (L) was prepared by treating L-cysteine methyl ester hydrochloride with ethyl oxalyl chloride in dichloromethane in the presence of triethylamine.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: D J Robins
• Keywords: Organic chemistry
• Date of Award: 1992
• Depositing User: Enlighten Team
• Unique ID: glathesis:1992-75018
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 13 Nov 2019 15:58
• Last Modified: 13 Nov 2019 15:58
• URI: https://theses.gla.ac.uk/id/eprint/75018