Thiamin phosphate synthase

The Thil gene, which encodes aother sulfurtransferase protein, is also needed.3743 The enzymology of the insertion of this sulfur into the thiazole is uncertain but may resemble that involved in synthesis of biotin, lipoic acid, and molybdopterin.374 Linkage of the two parts of the thiamin molecule (step d, Fig. 25-21) is catalyzed by thiamin phosphate synthase, evidently via an SN2 type reaction.377-37713... 

Khare G, Kar R, Tyagi AK (2011) Identification of inhibitors against Mycobacterium tuberculosis thiamin phosphate synthase, an important target for the development of anti-TB drugs. PLoS One 6 e22441... 

The thiamin phosphate synthase-catalyzed formation of thiamin phosphate from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 4-methyl-5-( 1 -hydroxyethyl)thiazole phosphate has been studied. A mechanism was proposed, and the substituent effects of the pyrimidine ring upon the TS discussed <2001B10095>. 

Vitamin Bi is an essential co-factor for several enzymes of carbohydrate metabolism such as transketolase, pyruvate dehydrogenase (PDH), pyruvate decarboxylase and a-ketoglutarate dehydrogenase. To become the active co-factor thiamin pyrophosphate (TPP), thiamin has to be salvaged by thiamin pyrophosphokinase or synthesized de novo. In Escherichia coli and Saccharomyces cerevisiae thiamin biosynthesis proceeds via two branches that have to be combined. In the pyrimidine branch, 4-amino-5-hydroxymethy-2-methylpyrimidine (PIMP) is phosphorylated to 4-amino-2-methyl-5-hydroxymethyl pyrimidine diphosphate (PIMP-PP) by the enzyme HMP/HMP-P kinase (ThiD) however, the step can also be catalyzed by pyridoxine kinase (PdxK), an enzyme also responsible for the activation of vitamin B6 (see below). The second precursor of thiamin biosynthesis, 5-(2-hydroxyethyl)-4-methylthiazole (THZ), is activated by THZ kinase (ThiM) to 4-methyl-5-(2-phosphoethyl)-thiazole (THZ-P), and then the thia-zole and pyrimidine moieties, HMP-PP and THZ-P, are combined to form thiamin phosphate (ThiP) by thiamin phosphate synthase (ThiE). The final step, pyrophosphorylation, yields TPP and is carried out by thiamin pyrophosphorylase (TPK). 

Thiamin phosphate synthase has broader substrate tolerance than initially assumed and catalyzes the formation of thiamin phosphate not only from 4 and 12 but also from 14 (Figure 4, T. P. Begley, unpublished data). Thiazole 14 is the product of the bacterial thiazole synthase. It is tautomerized to 4 in a reaction... 

Figure 3 Structure of the pyrimidine carbocation 13 trapped at the active site of thiamin phosphate synthase. The C-0 and C-N bonds in the substrate and product are 1.44 and 1.57 A, respectiveiy.

Figure 4 Alternative substrates of thiamin phosphate synthase.

This thermodynamic driving force is particularly useful tvith multienzyme equilibrium systems such as that used in the gram-scale synthesis of tv ro equivalents ofo-xylulose 5-phosphate (104) from (26) (Figure 10.38) [171,172]. Similarly, the corresponding 1-deoxy-D-xylulose 5-phosphate tvas efficiently produced from pyruvate and (34) by the catalytic action of the thiamine diphosphate-dependent 1-deoxy-D-xylulose 5-phosphate synthase (DXS) (EC 2.2.1.7) from E. coli [173]. 

At the beginning of the MEP pathway, the glycolytic products, pyruvate and D-glyceraldehyde (GAP), are condensed in a transketolase reaction to deoxy-xylulose phosphate (DXP) by the deoxy-xylulose phosphate synthase (DXS) enzyme. DXP is the precursor for other pathways leading to pyridoxal and thiamine. 

Sprenger, G.A. et al.. Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol, Proc. Natl. Acad Sci. USA 94, 12857, 1997. Lange, B.M. et al., A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway, Proc. Natl. Acad Sci. USA 95, 2100, 1998. Lois, L.M. et al., Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1- deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis, Proc. Natl. Acad. Sci. USA 95, 2105, 1998. 

The CPPase substrate DMAPP (15) is formed from isopentenyl pyrophosphate (IPP) (14) via the IPP isomerase reaction. It had been assumed that IPP was generated only via mevalonic acid (12) (Fig. 2), but Rohmer discovered another route, 2-C-methyl-D-erythritol 4-phosphate (13) (MEP) pathway (Fig. 2) [22, 23]. A key step in the MEP pathway is the reaction catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which combines hydroxyethyl thiamine pyrophosphate (hydroxyethyl TPP) generated from pyruvic acid (17) and TPP with glyceral-dehyde 3-phosphate (18) to yield 1-deoxy-D-xylulose 5-phosphate (19) containing five carbons. The mevalonate pathway operates in the cytosol of plants and animals, whereas the MEP pathway is present in the plastid of plants or in eubacteria [24-27]. 

Sprenger, G.A., Schorken, U., Wiegert, T. et cd. (1997) Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1 -deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, andpyridoxol. Proc. Natl. Acad. Sci. USA, 94, 12857-12862. 

The terpenes, carotenoids, steroids, and many other compounds arise in a direct way from the prenyl group of isopentenyl diphosphate (Fig. 22-1).16a Biosynthesis of this five-carbon branched unit from mevalonate has been discussed previously (Chapter 17, Fig. 17-19) and is briefly recapitulated in Fig. 22-1. Distinct isoenzymes of 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) in the liver produce HMG-CoA destined for formation of ketone bodies (Eq. 17-5) or mevalonate.7 8 A similar cytosolic enzyme is active in plants which, collectively, make more than 30,000 different isoprenoid compounds.910 However, many of these are formed by an alternative pathway that does not utilize mevalonate but starts with a thiamin diphosphate-dependent condensation of glyceraldehyde 3-phosphate with pyruvate (Figs. 22-1,22-2). 

In a very imaginative piece of research Frost and coworkers have developed a plasmid-based method for synthesizing aromatic amino acids, by incorporating the genes that code for the enzymes that perform the series of conversions from D-fructose-6-phosphate to D-erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonic acid-7-phos-phate (DAHP) near each other on a plasmid that can be transformed in E. coli. The enzymes are the thiamin diphosphate-dependent enzyme transketolase in the nonoxida-tive pentose shunt and DAHP synthase. The DAHP is then converted to the cyclic dehydroquinate, a precursor to all aromatic amino acids L-Tyr, L-Phe and L-Trp165,166 (equation 27). 

As already seen in Schemes 11.28 (pyruvate to ethanol) and 11.30 (pyruvate to acetyl-CoA), the three-carbon unit of pyruvate is a good substrate for reaction with the thiamine diphosphate cofactor. Indeed, as shown in Scheme 11.42, the same two-carbon unit found in Scheme 11.30 is generated, but now, in place of a lipoic acid fragment, attack occurs on the carbonyl of glyceraldehyde 3-phosphate under the influence of l-deoxy-o-xylulose synthase (EC 2.2.1.7). 

Scheme 11.42. A representation of the formation of 1-deoxy-D-xylulose phosphate from the reaction of thiamine diphosphate yUd with pyruvic acid and D-glyceraldehyde 3-phosphate in the presence of the synthase EC 2.2.I.7.

The 2,3-butanediol cycle in bacteria, as proposed by Juni and Heym (1956). The boxed area represents 2,3-BD formation in bacteria. BDL butanediol TPP thiamine pyrophosphate AACS acetyiacetoin synthase AACR acetyiacetoin reductase NAD nicotinamide adenine dinucleotide NADH reduced nicotinamide adenine dinucleotide NADP nicotinamide adenine dinucleotide phosphate NADPH reduced nicotinamide adenine dinucleotide phosphate (Redrawn from Syu (2001), with permission)... 

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