Glucose diphosphate formation

From extensive analysis of recombinant proteins, and the crystal structure of A. thaliana protein, detailed reaction mechanisms have been proposed. The ANS reaction likely proceeds via stereospecific hydroxylation of the leucoanthocyanidin (flavan-3,4-cA-diol) at the C-3 to give a flavan-3,3,4-triol, which spontaneously 2,3-dehydrates and isomerizes to 2-flaven-3,4-diol, which then spontaneously isomerizes to a thermodynamically more stable anthocyanidin pseudobase, 3-flaven-2,3-diol (Figure 3.2). The formation of 3-flaven-2,3-diol via the 2-flaven-3,4-diol was previously hypothesized by Heller and Forkmann. The reaction sequence, and the subsequent formation of the anthocyanidin 3-D-glycoside, does not require activity of a separate dehydratase, which was once postulated. Recombinant ANS and uridine diphosphate (UDP)-glucose flavonoid 3-D-glucosyltransferase (F3GT, sometimes... 

In the first Section, the dolichol pathway of protein glycosylation is introduced, and the reader is made familiar with the various reactions in the formation of the lipid and carbohydrate moieties of lipid-linked saccharides. Three different classes of compound are known so far (a) isoprenoid alcohol esters of monosaccharide monophosphates, such as D-mannosyl and D-glucosyl (dolichol phosphate), (b) such isoprenoid alcohol esters of saccharide diphosphates as dolichol diphosphate linked to 2-acetamido-2-deoxy-D-glucose and to oligosaccharides, and (c) retinol (D-mannosyl phosphate). The dolichol-linked sugars occur in all eukaryotes. 

Enzyme poisons have been studied mainly in attempts to understand the normal physiological processes. As would be expected, synthesis and accumulation of sucrose have often been drastically lowered. In particular, the inhibition of the formation of D-fructose diphosphate and the conversion of D-glucose into D-fructose inhibited the formation of sucrose.288 330... 

Colvin155 was the first to postulate a lipid-bound D-glucose as an intermediate in the biosynthesis of bacterial cellulose. Lipid-sugar derivatives, tentatively identified as lipid-diphosphate-D-glucose, lipid-diphosphate-cellobiose, and, perhaps, higher polymers, were detected in this system.128 These lipid-sugar compounds, which were acid- and alkali-labile, seemed to be formed prior to cellulose, and their formation was inhibited by adding... 

O. Gabriel and G. Ashwell, Biological mechanisms involved in the formation of deoxysugars. I. Preparation of thymidine diphosphate glucose labelled specifically in carbon 3, J. Biol. Chem., 240 (1965) 4123-4127. 

However, nucleoside diphosphates (NDP) are still expensive substrates, which can be obtained from much more cheaper nucleoside monophosphates (NMP). In this respect we have combined the SuSy-catalyzed cleavage of sucrose with the enzymatic formation of NDPs from NMPs catalyzed by nucleoside monophosphate kinase (NMPK, EC 2.7.4.4) or myokinase (MK, EC 2.7.4.3), including in situ regeneration of ATP with pyruvate kinase (PK, EC 2.7.1.40) (Fig. 20) [272]. Testing the substrate spectrum of four different kinases disclosed that none of them accepted dTMP as substrate [272], However, dUMP was well accepted by NMPK and dUDP-activated glucose could also substitute dTDP-activated glucose as precursor for the synthesis of activated deoxysugars (see below). The excellent enzyme stabilities under synthesis... 

Phosphoglucomutase (EC 2.7 5.l) slowly converts R-l-P to ribose-5-phosphate. A mixture of 35 mM R-l-f O J-P, 17mM R-l-[ L 0l(]-P and less than lmg glucose-1,6-diphosphate at pH 7 33 was equilibrated with phosphoglucomutase (Sigma, P3397, rabbit muscle), at 25°C for 3 Hr. The 31p NMR spectrum was recorded at pH 7 37- The enzyme converted ca. 20 to a mixture of ribose-5-phosphate l Ol (resonance identified by addition of authentic material) and I80I803 species. This demonstrated that the enzyme catalyzed formation of the 0-P bond. 

Fig. 3-2. Formation of uridine diphosphate glucose (UDP-D-glucose) (3) from a-D-glucopyranosyl-1-phosphate (1) and uridine triphosphate (2) under simultaneous release of pyrophosphate (4).

Fig. 3-4. Simplified representation of the formation of hemicellulose precursors from UDP-d-glucose or GDP-D-glucose. Note that NDP (nucleotide diphosphate) means either UDP or GDP.

Lavintman, N., Tandecarz, J., Carceller, M., Mendiara, S., and Cardini, C. E. 1974. Role of uridine diphosphate glucose in the biosynthesis of starch Mechanism of formation and enlargement of a glucoproteic acceptor. Eur. J. Biochem. 50,145-155. 

Fig. 2. Reactions catalyzed by galactosyltransferase (GT). (a) The incorporation of galactose (Gal) into a (1—>4) linkage with JV-acelylglucosamine (NAG) to form N-acetyl-lactosamine (NAL). UDP, Uridine diphosphate, (b) Modification of the activity of GT by a-lactalbumin (a-LA) to convert it to a lactose synthase catalyzing the formation of lactose from UDP-Gal and glucose.

---