This thesis describes studies on the last two enzymes on the biotin biosynthetic pathway. The penultimate enzyme, dethiobiotin synthetase (DTBS), catalyses the <I>ureido</I> ring closure of dethiobiotin from (7<I>R,</I>8<I>S</I>)-7,8-diaminononanoate (DAPA), carbon dioxide and ATP. The final step, catalysed by biotin synthase, involves the chemically difficult insertion of sulphur into the unacativated carbon skeleton of dethiobiotin to yield biotin. A range of analogues of DAPA differing in chain length and C-8 substitution were synthesised and found to act as slow substrates of DTBS. Steady-state kinetic and binding parameters were evaluated for these modified substrates and their relevance to the mechanism of the DTBS reaction is discussed. Crystallographic and modelling studies of one analogue bound in the active site of DTBS suggest that the enzyme may employ an alternative mechanism of <I>ureido</I> ring closure to that of the native substrate. Biotin production was demonstrated in cell-free extracts of an <I>E. coli bio</I>B transformant and the origin of the sulphur atom in biotin was investigated. A novel cysteine desulphurase enzyme, which catalyses the of L-cysteine to give L-alanine and sulphur, was discovered and its possible role in the biotin synthase reaction is discussed. [6,6-<SUP>2</SUP>H<SUB>2</SUB>]-(±)-dethiobiotin and [6,6-F<SUB>2</SUB>]-(±)-dethiobiotin were identified as target compounds in intermediate trapping experiments and as selective biocides. A synthetic route to these compounds was devised and the introduction of label was explored using labelled catalysts and carbonyl transformation chemistry.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:657127 |
| Date | January 1995 |
| Creators | McPherson, Michael J. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/11153 |
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