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Studies of thiamine biosynthesis

Studies of thiamine biosynthesis
Studies of thiamine biosynthesis
In Escherichia coli, and other prokaryotes, thiamine (vitamin B1) is assembled by coupling 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (Hmp-PP) and 4-methyl-5-(?-hydroxyethyl)thiazole phosphate (Thz-P). The thiazole moiety is biosynthesised from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine, and at least six genes are required including thiH, thiG, thiS, and thiF. Whilst in aerobes, the C2-N3 fragment of Thz-P derives from glycine in a reaction catalysed by the flavoenzyme ThiO, in anaerobes such as E. coli, dehydroglycine is formed from tyrosine in a ThiH dependent reaction. This biosynthetic step requires the cleavage of the C?-C? bond of tyrosine and a release of an aromatic side chain. ThiH shows sequence similarity with the ‘radical S-adenosylmethionine’ (AdoMet) family of proteins, including conserved cysteine ligands to the essential [4Fe-4S] cluster and has been shown to form a complex with ThiG. With the purpose of studying the mechanistic enzymology by which Thz-P is assembled it was crucial to isolate ThiH in the holo-form. Several expression systems and purification methodologies were investigated. The optimisation of the purification method, together with in vitro chemical reconstitution with exogenous iron and sulfide allowed the successful isolation of holo-ThiH.
To facilitate the mechanistic investigation of Thz-P biosynthesis, an in vitro assay was developed, and the reaction products formed in vitro were elucidated and quantified. The aromatic by-product derived from the side chain of tyrosine is p-cresol and the remaining fragment yields glyoxylate, a product of hydrolysis of dehydroglycine
Martins, Filipa Teixeira
b308f776-164f-425b-8bc6-84d7c9bf3052
Martins, Filipa Teixeira
b308f776-164f-425b-8bc6-84d7c9bf3052
Roach, Peter
ca94060c-4443-482b-af3e-979243488ba9

Martins, Filipa Teixeira (2009) Studies of thiamine biosynthesis. University of Southampton, School of Chemistry, Doctoral Thesis, 227pp.

Record type: Thesis (Doctoral)

Abstract

In Escherichia coli, and other prokaryotes, thiamine (vitamin B1) is assembled by coupling 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (Hmp-PP) and 4-methyl-5-(?-hydroxyethyl)thiazole phosphate (Thz-P). The thiazole moiety is biosynthesised from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine, and at least six genes are required including thiH, thiG, thiS, and thiF. Whilst in aerobes, the C2-N3 fragment of Thz-P derives from glycine in a reaction catalysed by the flavoenzyme ThiO, in anaerobes such as E. coli, dehydroglycine is formed from tyrosine in a ThiH dependent reaction. This biosynthetic step requires the cleavage of the C?-C? bond of tyrosine and a release of an aromatic side chain. ThiH shows sequence similarity with the ‘radical S-adenosylmethionine’ (AdoMet) family of proteins, including conserved cysteine ligands to the essential [4Fe-4S] cluster and has been shown to form a complex with ThiG. With the purpose of studying the mechanistic enzymology by which Thz-P is assembled it was crucial to isolate ThiH in the holo-form. Several expression systems and purification methodologies were investigated. The optimisation of the purification method, together with in vitro chemical reconstitution with exogenous iron and sulfide allowed the successful isolation of holo-ThiH.
To facilitate the mechanistic investigation of Thz-P biosynthesis, an in vitro assay was developed, and the reaction products formed in vitro were elucidated and quantified. The aromatic by-product derived from the side chain of tyrosine is p-cresol and the remaining fragment yields glyoxylate, a product of hydrolysis of dehydroglycine

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Published date: September 2009
Organisations: University of Southampton

Identifiers

Local EPrints ID: 71854
URI: http://eprints.soton.ac.uk/id/eprint/71854
PURE UUID: 05b6883e-fa51-4aec-b7c9-dac2e5b8338f
ORCID for Peter Roach: ORCID iD orcid.org/0000-0001-9880-2877

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Date deposited: 20 Jan 2010
Last modified: 30 Jan 2020 01:30

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