焦磷酸硫胺
化学
生物化学
生物合成
酪氨酸
半胱氨酸
噻唑
立体化学
硫胺素
氨基酸
芳香族氨基酸
辅因子
酶
摘要
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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