Branching enzyme isolation

In nature a cascade of enzyme-catalyzed reactions is involved for the biosynthesis of starch. When selecting the appropriate enzymes and reaction circumstances reactions with multiple enzymes can be performed in vitro. Synthetic glycogen was first synthesized in vitro by Cori [146] in 1943 via the cooperative action of muscle phosphorylase and branching enzymes isolated from rat liver and rabbit heart. 

Dihydroxyacid dehydratase of the branched-chain amino acid biosynthetic pathway catalyzes the dehydration and tautomerization of 2,3-dihydroxy-3-methyl-(butyrate and pentanoate) to 2-keto-3-methyl(butyrate and pentanoate). The enzyme isolated from spinach recently has been shown to have not a [4Fe-4S] cluster, but rather a spectroscopically unusual [2Fe-2S] cluster in its active site (68,69). The EPR spectrum of the reduced enzyme is similar to that seen for Rieske Fe-S proteins (71) with a g-average of 1.91. Upon addition of substrate the g-average of the EPR spectrum shifts to 1.96 (opposite the effect of substrate on aconitase), and then reverts back to a g-average of 1.90 when only the product is present The dramatic changes in the EPR spectra upon addition of substrate suggest, in analogy to aconitase, that the Fe-S cluster may be directly involved in catalysis. 

Representatives of all kinds have been explored for synthetic applications while mechanistic investigations were mainly focussed on the distinct FruA enzymes isolated from rabbit muscle [196] and yeast [197,198]. For mechanistic reasons, all DHAP aldolases appear to be highly specific for the donor component DHAP [199], and only a few isosteric replacements of the ester oxygen for sulfur (46), nitrogen (47), or methylene carbon (48) were found to be tolerable in preparative experiments (Fig. 7) [200,201], Earlier assay results [202] that had indicated activity also for a racemic methyl-branched DHAP analog 53 are now considered to be artefactual [203]. Dihydroxyacetone sulfate 50 has been shown to be covalently bound via Schiff base formation, but apparently no a-deprotonation occurred as neither H/D-exchange nor C-C... 

Phytoglycogen-branching enzyme has been found in du however, no phytoglycogen was isolated by Black et al.23 Preiss and Boyer396 reported that the du mutation lowered starch synthase II activity and also lowered branching enzyme Ha activity. Gao et al.397 used a molecular approach to clone the du gene in maize endosperms and, based on amino acid sequence similarity of the predicted protein product with the soluble starch synthase III of potato,398 concluded that du most likely encodes the 180 000 molecular weight, primer-dependent soluble starch synthase described previously.399,400,401... 

Isolation of cDNA Clones Encoding the Branching Enzyme Isozyme Genes... 

Few studies on the localization of the starch biosynthetic enzymes were done before 1978, when it was found that ADPGlc PPase was located exclusively in the chloroplast fraction in both spinach (Mares et al., 1978) and pea (Levi and Preiss, 1978). The first detailed study was done by Okita et al. (1979), in which spinach leaf chloroplasts were isolated either by differential centrifugation (Walker, 1971 see also later) or from protoplasts (Nishimura et al, 1976). These plastid preparations contained essentially all of the activity of the starch biosynthetic enzymes, ADPGlc PPase, starch synthase, and branching enzyme. Subsequently, in guard cells of Commelina communis, Robinson and Preiss (1987) showed that the starch biosynthetic enzymes were present exclusively in the chloroplast fraction. 

To produce amylopectin, it is necessary to have a starch branching enzyme that is capable of introducing the a-(1 6) branch linkages into the linear amylose molecules synthesized by starch synthase. Such an enzyme (Q-enz)mre) was first identified in potatoes [131] and then in broad beans [132]. The potato Q-enzyme has been isolated and purified and the properties determined [133]. Q-enzyme is a glucanyltransferase and cleaves an a-jl— 4) linkage of an amylose chain, and then transfers the chain to the C6-OH of another amylose-chain, rather than being transferred and attached to the residual part of the cleaved chain [134]. It was shown that water is not involved in these branching reactions [133]. 

Kitahata, S., and S. Okada, Branching enzymes, in Handbook of Amylase and Related Enzymes. Their Sources, Isolation Methods, Properties and Applications, The Amylase Research Society of Japan, Ed. Pergamon Press, Oxford, U.K., 1988, pp. 143-154. 

Both described enzymes are isolated from natural sources. Phosphorylase is isolated from potatoes whereas the glycogen branching enzyme is produced by various bacterial sources. Depending on the source the properties of the products and the reaction conditions may differ. 

Mukerjea, R., Falconer, D.X, Yoon, S.H., and Robyt, J.F. 2010. Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes starch synthase and branching enzyme from potato tubers. Carbohydrate Research, 345, (11), 1555-1563. 

The reformed glycogen was then isolated and incubated with the purified transglucosidase. If the action of the branching enzyme consisted in the shifting of chain segments by transglucosidation, the new structure... 

V. J. Thon, M. Khalil, J. F. Cannon, Isolation of human glycogen branching enzyme cDNAs by screening complementation in yeast J. Biol Chem. 268 1993, 7509-13. 

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