Challand, MR, Martins, FT and Roach, PL (2010) Catalytic activity of the anaerobic tyrosine lyase required for thiamine biosynthesis in Escherichia coli. J. Biol. Chem. 285:5240-8
Thiazole synthase in Escherichia coli is an alphabeta heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the C alpha-C beta 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 alpha-C beta 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.
Anaerobiosis/physiology; Carbon-Carbon Lyases/antagonists & inhibitors; Carbon-Carbon Lyases/chemistry; Carbon-Carbon Lyases/metabolism; Catalysis; Cresols/chemistry; Cresols/metabolism; Deoxyadenosines/chemistry; Deoxyadenosines/metabolism; Enzyme Inhibitors/chemistry; Escherichia coli/enzymology; Escherichia coli Proteins/antagonists & inhibitors; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Kinetics; S-Adenosylmethionine/chemistry; S-Adenosylmethionine/metabolism; Substrate Specificity; Tyrosine/chemistry; Tyrosine/metabolism
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