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Mechanistic aspects of thiamine biosynthesis in escherichia coli

Mechanistic aspects of thiamine biosynthesis in escherichia coli
Mechanistic aspects of thiamine biosynthesis in escherichia coli
The biosynthesis of the vitamin thiamine is laden with interesting chemistry. In anaerobic bacteria (such as Escherichia coli) a reactive intermediate, dehydroglycine, required for the biosynthesis of the thiazole moiety is derived from cleavage of the C??C? bond of tyrosine in a reaction catalysed by the radical S-adenosyl-L-methionine (SAM) enzyme, tyrosine lyase (ThiH). The aromatic by-product of this reaction in vivo was unequivocally characterised as p-cresol, a result which supported previous in vitro observations. Development of a reproducible activity assay for ThiH activity initiated detailed studies on the kinetics. ThiH, isolated either in a complex with thiazole synthase (ThiG), or as a monomer displayed pre-steady state burst phase kinetics. The SAM cleavage products (5‘-deoxyadenosine (DOA) and methionine) were identified as inhibitors of ThiH activity and this inhibition could be alleviated by addition of 5‘-methylthioadenosine / S-adenosylhomocysteine nucleosidase (MTAN), which catalysed rapid hydrolysis of DOA to adenine and 5‘-deoxyribose. The addition of MTAN increased the activity of the ThiGH complex but drastically increased the amount of observed uncoupled cleavage of SAM. The in vitro products of tyrosine cleavage (glyoxylate and ammonium ions) were also identified as inhibitors of the ThiH mediated tyrosine cleavage reaction. However, reductive cleavage of SAM continued in an uncoupled manner. Experiments with tyrosine analogues showed that 4-hydroxyphenylpropionic acid compounds could support uncoupled SAM cleavage, but the C??C? bond cleavage reaction was dependent on a correctly orientated ?-amino functional group. A mechanistic model was constructed, based on the available experimental data, which explained the observed product inhibition, uncoupled turnover and complex formation between ThiH and ThiG. The model proposes that ThiH controls the tyrosine cleavage reaction in order to co-ordinate the generation of the intermediate, iv dehydroglycine, with its utilisation in the thiazole forming cylisation reaction, catalysed by ThiG
Challand, M.R
e887b78b-7545-4c64-846a-ade3be4e6b99
Challand, M.R
e887b78b-7545-4c64-846a-ade3be4e6b99
Roach, Peter
ca94060c-4443-482b-af3e-979243488ba9

Challand, M.R (2011) Mechanistic aspects of thiamine biosynthesis in escherichia coli. University of Southampton, Chemistry, Doctoral Thesis, 220pp.

Record type: Thesis (Doctoral)

Abstract

The biosynthesis of the vitamin thiamine is laden with interesting chemistry. In anaerobic bacteria (such as Escherichia coli) a reactive intermediate, dehydroglycine, required for the biosynthesis of the thiazole moiety is derived from cleavage of the C??C? bond of tyrosine in a reaction catalysed by the radical S-adenosyl-L-methionine (SAM) enzyme, tyrosine lyase (ThiH). The aromatic by-product of this reaction in vivo was unequivocally characterised as p-cresol, a result which supported previous in vitro observations. Development of a reproducible activity assay for ThiH activity initiated detailed studies on the kinetics. ThiH, isolated either in a complex with thiazole synthase (ThiG), or as a monomer displayed pre-steady state burst phase kinetics. The SAM cleavage products (5‘-deoxyadenosine (DOA) and methionine) were identified as inhibitors of ThiH activity and this inhibition could be alleviated by addition of 5‘-methylthioadenosine / S-adenosylhomocysteine nucleosidase (MTAN), which catalysed rapid hydrolysis of DOA to adenine and 5‘-deoxyribose. The addition of MTAN increased the activity of the ThiGH complex but drastically increased the amount of observed uncoupled cleavage of SAM. The in vitro products of tyrosine cleavage (glyoxylate and ammonium ions) were also identified as inhibitors of the ThiH mediated tyrosine cleavage reaction. However, reductive cleavage of SAM continued in an uncoupled manner. Experiments with tyrosine analogues showed that 4-hydroxyphenylpropionic acid compounds could support uncoupled SAM cleavage, but the C??C? bond cleavage reaction was dependent on a correctly orientated ?-amino functional group. A mechanistic model was constructed, based on the available experimental data, which explained the observed product inhibition, uncoupled turnover and complex formation between ThiH and ThiG. The model proposes that ThiH controls the tyrosine cleavage reaction in order to co-ordinate the generation of the intermediate, iv dehydroglycine, with its utilisation in the thiazole forming cylisation reaction, catalysed by ThiG

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Published date: 1 April 2011
Organisations: University of Southampton

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Local EPrints ID: 191353
URI: https://eprints.soton.ac.uk/id/eprint/191353
PURE UUID: 372cdf6a-3939-4f7d-81e6-0389161a9ae0
ORCID for Peter Roach: ORCID iD orcid.org/0000-0001-9880-2877

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Date deposited: 21 Jun 2011 07:47
Last modified: 14 Aug 2018 00:34

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