Facile enzymic de novo synthesis and NMR spectroscopic characterization of D-tagatose 1,6-bisphosphate

Article abstract of DOI:10.1016/0008-6215(93)87020-S

A D-tagatose 1,6-bisphosphate aldolase requiring Zn(2+) for catalytic activity (class II) was purified from E.coli cells grown on galactitol.The aldolase, a homotetramer composed of subunits of mol wt ca. 28000, had a pH optimum at 7.5 and was highly selective for L-erythro as compared to D-threo stereochemistry (99:1).This allowed its application in a coupled enzyme system together with glycerol kinase, pyruvate kinase and triose phosphate isomerase for the de novo, one-pot synthesis of D-tagatose 1,6-bisphosphate starting from dihydroxyacetone and phosphoenolpyruvate (for the in situ regeneration of adenosine triphosphate), in quantities of 10 mmol.The expeditious process compares very favorably in simplicity and yield (40percent overall) with the known multistep chemical preparation even after improvements to the latter accomplished during the present work.The classical sequence, which starts from D-galacturonic acid, was modified at both phosphorylation steps: 1,2:3,4-di-O-isopropylidene-D-tagatofuranose was esterified by application of the trivalent phosphitylation agent dibenzyl di-N-ethyl-phosphoramidite followed by hydrogen peroxide oxidation, and a bacterial fructose 6-phosphate kinase was used for enzymic phosphorylation of D-tagatose 6-phosphate.For the latter enzyme, which was the isoenzyme Pfk-2 from a recombinant strain of E. coli, kinetic constants were determined.NMR spectroscopic assignments are presented for D-tagatose and its phosphates.