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Catalytic activity of the anaerobic tyrosine lyase required for thiamine biosynthesis in Escherichia coli

Catalytic activity of the anaerobic tyrosine lyase required for thiamine biosynthesis in Escherichia coli
Catalytic activity of the anaerobic tyrosine lyase required for thiamine biosynthesis in Escherichia coli
Thiazole synthase in Escherichia coli is an ?? heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the C?–C? bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cyclization reaction catalyzed by ThiG. ThiH is a radical S-adenosylmethionine (AdoMet) enzyme that utilizes a [4Fe-4S]+ cluster to reductively cleave AdoMet, forming methionine and a 5?-deoxyadenosyl radical. Analysis of the time-dependent formation of the reaction products 5?-deoxyadenosine (DOA) and p-cresol has demonstrated catalytic behavior of the tyrosine lyase. The kinetics of product formation showed a pre-steady state burst phase, and the involvement of DOA in product inhibition was identified by the addition of 5?-methylthioadenosine/S-adenosylhomocysteine nucleosidase to activity assays. This hydrolyzed the DOA and changed the rate-determining step but, in addition, substantially increased the uncoupled turnover of AdoMet. Addition of glyoxylate and ammonium inhibited the tyrosine cleavage reaction, but the reductive cleavage of AdoMet continued in an uncoupled manner. Tyrosine analogues were incubated with ThiGH, which showed a strong preference for phenolic substrates. 4-Hydroxyphenylpropionic acid analogues allowed uncoupled AdoMet cleavage but did not result in further reaction (C?–C? bond cleavage). The results of the substrate analogue studies and the product inhibition can be explained by a mechanistic hypothesis involving two reaction pathways, a product-forming pathway and a futile cycle.
enzymes/inhibitors, enzymes/kinetics, enzymes/mechanisms, protein/iron-sulfur, radicals, vitamins and cofactors/vitamin b, product inhibition
0021-9258
5240-5248
Challand, M.R
e887b78b-7545-4c64-846a-ade3be4e6b99
Martins, F.T.
77b09009-d0c9-4afc-b3b3-ea318cb24fc4
Roach, P.L.
ca94060c-4443-482b-af3e-979243488ba9
Challand, M.R
e887b78b-7545-4c64-846a-ade3be4e6b99
Martins, F.T.
77b09009-d0c9-4afc-b3b3-ea318cb24fc4
Roach, P.L.
ca94060c-4443-482b-af3e-979243488ba9

Challand, M.R, Martins, F.T. and Roach, P.L. (2010) Catalytic activity of the anaerobic tyrosine lyase required for thiamine biosynthesis in Escherichia coli. The Journal of Biological Chemistry, 285 (8), 5240-5248. (doi:10.1074/jbc.M109.056606). (PMID:19923213)

Record type: Article

Abstract

Thiazole synthase in Escherichia coli is an ?? heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the C?–C? bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cyclization reaction catalyzed by ThiG. ThiH is a radical S-adenosylmethionine (AdoMet) enzyme that utilizes a [4Fe-4S]+ cluster to reductively cleave AdoMet, forming methionine and a 5?-deoxyadenosyl radical. Analysis of the time-dependent formation of the reaction products 5?-deoxyadenosine (DOA) and p-cresol has demonstrated catalytic behavior of the tyrosine lyase. The kinetics of product formation showed a pre-steady state burst phase, and the involvement of DOA in product inhibition was identified by the addition of 5?-methylthioadenosine/S-adenosylhomocysteine nucleosidase to activity assays. This hydrolyzed the DOA and changed the rate-determining step but, in addition, substantially increased the uncoupled turnover of AdoMet. Addition of glyoxylate and ammonium inhibited the tyrosine cleavage reaction, but the reductive cleavage of AdoMet continued in an uncoupled manner. Tyrosine analogues were incubated with ThiGH, which showed a strong preference for phenolic substrates. 4-Hydroxyphenylpropionic acid analogues allowed uncoupled AdoMet cleavage but did not result in further reaction (C?–C? bond cleavage). The results of the substrate analogue studies and the product inhibition can be explained by a mechanistic hypothesis involving two reaction pathways, a product-forming pathway and a futile cycle.

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More information

Published date: 19 February 2010
Keywords: enzymes/inhibitors, enzymes/kinetics, enzymes/mechanisms, protein/iron-sulfur, radicals, vitamins and cofactors/vitamin b, product inhibition
Organisations: Chemistry

Identifiers

Local EPrints ID: 146147
URI: https://eprints.soton.ac.uk/id/eprint/146147
ISSN: 0021-9258
PURE UUID: 5bc8aa7b-f267-4813-8264-689d895477eb
ORCID for P.L. Roach: ORCID iD orcid.org/0000-0001-9880-2877

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Date deposited: 30 Jun 2010 13:47
Last modified: 20 Jul 2019 01:07

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Contributors

Author: M.R Challand
Author: F.T. Martins
Author: P.L. Roach ORCID iD

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