Maltose metabolism in the hyperthermophilic archaeon Thermococcus litoralis: Purification and characterization of key enzymes

Maltose metabolism was investigated in the hyperthermophilic archaeon Therm ococcus litoralis. Maltose was degraded by the concerted action of 4-alpha- glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than fou r, to produce glucose-l-phosphate. Phosphoglucomutase activity was also det ected in T. litoralis cell extracts. Glucose derived from the action of 4- alpha-glucanotransferase was subsequently metabolized via an Embden-Meyerho f pathway. The closely related organism Pyrococcus furiosus used a differen t metabolic strategy in which maltose was cleaved primarily by the action o f an alpha-glucosidase, a p-nitrophenyl-alpha-D-glucopyranoside (PNPG)-hydr olyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for thi s enzyme. The two key enzymes in the pathway for maltose catabolism in T. l itoralis were purified to homogeneity and characterized; they were constitu tively synthesized, although phosphorylase expression was twofold induced b y maltodextrins or maltose. The gene encoding MalP was obtained by compleme ntation in Escherichia coli and sequenced (calculated molecular mass, 96,62 2 Da). The enzyme purified from the organism had a specific activity for ma ltoheptaose, at the temperature for maximal activity (98 degrees C), of 66 U/mg. AK(m) of 0.46 mM was determined with heptaose as the substrate at 60 degrees C. The deduced amino acid sequence had a high degree of identity wi th that of the putative enzyme from the hyperthermophilic archaeon Pyrococc us horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthe rmophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosi s (31%) but not with that of the enzyme from E. coil (13%). The consensus b inding site for pyridoxal 5'-phosphate is conserved in the T. litoralis enz yme.