Copurification of hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase of Saccharomyces cerevisiae: characterization of hydroxyethylthiazole kinase as a bifunctional enzyme in the thiamine biosynthetic pathway

Kawasaki, Yuko

doi:10.1128/jb.175.16.5153-5158.1993

Cited by 28 publications

(14 citation statements)

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“…The inner cavity of the cage has a maximum diameter of about 55 Å (Figure 1C). The presence of the cavity contributes to the high solvent content (~60%) of the crystals, and explains previous results from analytical size exclusion chromatography, which suggested THI6 was an octamer (23). The caps of the prolate spheroid are formed by trimeric assemblies of the C-terminal (ThiM) domains and the equator is formed by dimeric assemblies of the N-terminal (TPS) domains.…”

Section: Resultssupporting

confidence: 78%

Domain Organization in Candida glabrata THI6, a Bifunctional Enzyme Required for Thiamin Biosynthesis in Eukaryotes,

et al. 2010

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THI6 is a bifunctional enzyme found in the thiamin biosynthetic pathway in eukaryotes. The Nterminal domain of THI6 catalyzes the ligation of the thiamin thiazole and pyrimidine moieties to form thiamin phosphate and the C-terminal domain catalyzes the phosphorylation of 4-methyl-5-hydroxyethylthiazole in a salvage pathway. In prokaryotes, thiamin phosphate synthase and 4-methyl-5-hydroxyethylthiazole kinase are separate gene products. Here we report the first crystal structure of a eukaryotic THI6 along with several complexes that characterize the active sites responsible for the two chemical reactions. THI6 from Candida glabrata is a homohexamer in which the six protomers form a cage-like structure. Each protomer is composed of two domains, which are structurally homologous to their monofunctional bacterial counterparts. Two loop regions not found in the bacterial enzymes provide interactions between the two domains. The structures of different protein-ligand complexes define the thiazole and ATP binding sites of the 4-methyl-5-hydroxyethylthiazole kinase domain, and the thiazole phosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate binding sites of the thiamin phosphate synthase domain. Our structural studies reveal that the active sites of the two domains are 40 Å apart and are not connected by an obvious channel. Biochemical studies show 4-methyl-5-hydroxyethylthiazole phosphate is a substrate for THI6; however, adenosine diphospho-5-β-ethyl-4-methylthiazole-2-carboxylic acid, the product of THI4, is not a substrate for THI6. This suggests that unidentified enzyme is necessary to produce the substrate for THI6 from the THI4 product.Thiamin (vitamin B 1 ) is an essential component of all living systems. The active form of thiamin, thiamin pyrophosphate (ThDP), acts as a cofactor for several important enzymes in carbohydrate and amino acid metabolism. The mechanistic role of the cofactor is stabilization of an acyl carbanion intermediate (1). Thiamin biosynthetic pathways are found in prokaryotes and some eukaryotes (yeast, fungi and plants); however, vertebrates cannot synthesize thiamin. Therefore, thiamin is an essential component of the human diet with a daily requirement of 1.0-1.2 mg (2). Deficiency of thiamin leads to diseases such as beriberi † This work was supported by NIH grant DK44083 to T.P.B. and DK67081 to S.E.E. || This work is based upon research conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines, which are supported by award RR-15301 from the National Center for Research Resources at the National Institutes of Health. Use of the Advanced Photon Source is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. ‡ The coordinates of the THI6 structures have been deposited in the Protein Data Bank under accession number 3NL2, 3NL3, 3NM3, 3NL5, 3NL6 and 3NM1 for THI6, THI6/ThMP, THI6/ThMP/PP, THI6/Thz/AMP-PCP, THI6/ThMP/AMP-PCP and THI6/CF 3 HMP-PP/carboxy-Thz-P, respec...

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Section: Resultssupporting

confidence: 78%

Domain Organization in Candida glabrata THI6, a Bifunctional Enzyme Required for Thiamin Biosynthesis in Eukaryotes,

et al. 2010

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“…The presumed upstream activating sequence, AAA(A/C)TCAA, is found in the 5Ј-upstream noncoding regions of PHO3 (3) and THI6 (37) genes by two copies for each gene. The THI6 gene product is a bifunctional enzyme with thiamin-phosphate pyrophosphorylase (EC 2.5.1.3) and 4-methyl-5-␤-hydroxyethylthiazole kinase (EC 2.7.1.50) activities (30). On the other hand, there is a TTGATTT sequence at positions 238 -244 of the THI10 5Ј-upstream noncoding region (Fig.…”

Section: Yeast Thiamin Transport Gene 19168mentioning

confidence: 99%

Isolation and Characterization of a Thiamin Transport Gene,THI10, from Saccharomyces cerevisiae

Enjo

Nosaka

Ogata

et al. 1997

Journal of Biological Chemistry

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Yeast cells take up thiamin from the extracellular environment by an active transport system with a pH optimum of 4.5 and a K m value of 0.18 M, and thiamin in the cells is concentrated ϳ10,000-fold over extracellular levels (1). The thiamin transport system is more specific for the chemical structure of the pyrimidine moiety than the thiazole moiety of thiamin (2). Saccharomyces cerevisiae also secretes a thiamin-repressible acid phosphatase encoded by PHO3 gene (3, 4) in a periplasmic space. Since yeast cells cannot take up thiamin phosphate esters, the PHO3 protein with a high affinity for thiamin phosphate esters appears to hydrolyze them before the uptake (5, 6), and then thiamin is converted to thiamin pyrophosphate (TPP) 1 by thiamin pyrophosphokinase (EC 2.7.6.2) encoded by the THI80 gene (7). TPP is an important cofactor in the energy metabolism (8) and is a negative effector of the regulation mechanism of thiamin metabolism in yeast cells (9).We report here the isolation and characterization of S. cerevisiae THI10 gene and provide evidence that the THI10 gene encodes for a thiamin transport carrier protein. The predicted THI10 protein is highly hydrophobic and shows significant sequence similarities to yeast uracil and allantoin transport proteins. The thiamin transport activity and the thiamin binding activity in the yeast plasma membrane fraction in thi10 null strain cells were restored when the THI10 open reading frame (ORF) was expressed by yeast GAL1 promoter. The regulation of the THI10 gene expression was also investigated.

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“…A natural bifunctional product of the THI6 gene was purified and characterized in the early nineties [28]. The two activities of the purified protein (EC 2.5.1.3 and EC 2.7.1.50, respectively) were inseparable, though their pH optima differed.…”

Section: Thiamin Phosphate Diphosphorylase/hydroxyethylthiazole Kinasmentioning

confidence: 99%

The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts

Kowalska

Kozik

2008

Cellular and Molecular Biology Letters

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Thiamin (vitamin B1) is an essential molecule for all living organisms. Its major biologically active derivative is thiamin diphosphate, which serves as a cofactor for several enzymes involved in carbohydrate and amino acid metabolism. Important new functions for thiamin and its phosphate esters have recently been suggested, e.g. in gene expression regulation by influencing mRNA structure, in DNA repair after UV illumination, and in the protection of some organelles against reactive oxygen species. Unlike higher animals, which rely on nutritional thiamin intake, yeasts can synthesize thiamin de novo. The biosynthesis pathways include the separate synthesis of two precursors, 4-amino -5-hydroxymethyl-2-methylpyrimidine diphosphate and 5-(2-hydroxyethyl)-4-methylthiazole phosphate, which are then condensed into thiamin monophosphate. Additionally, yeasts evolved salvage mechanisms to utilize thiamin and its dephosphorylated late precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole, from the environment. The current state of knowledge on the discrete steps of thiamin biosynthesis in yeasts is far from satisfactory; many intermediates are postulated only by analogy to the much better understood biosynthesis process in bacteria. On the other hand, the genetic mechanisms regulating thiamin biosynthesis in yeasts are currently under extensive exploration. Only recently, the structures of some of the yeast enzymes involved in thiamin biosynthesis, such as thiamin diphosphokinase and thiazole synthase, were determined at the atomic resolution, and mechanistic proposals for the catalysis of particular biosynthetic steps started to emerge.

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Copurification of hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase of Saccharomyces cerevisiae: characterization of hydroxyethylthiazole kinase as a bifunctional enzyme in the thiamine biosynthetic pathway

Cited by 28 publications

References 10 publications

Domain Organization in Candida glabrata THI6, a Bifunctional Enzyme Required for Thiamin Biosynthesis in Eukaryotes,

Domain Organization in Candida glabrata THI6, a Bifunctional Enzyme Required for Thiamin Biosynthesis in Eukaryotes,

Isolation and Characterization of a Thiamin Transport Gene,THI10, from Saccharomyces cerevisiae

The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts

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