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Journal of Bacteriology, June 1999, p. 3358-3367, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Maltose Metabolism in the Hyperthermophilic Archaeon Thermococcus litoralis: Purification and Characterization of Key Enzymes

Karina B. Xavier,1 Ralf Peist,2 Marina Kossmann,2 Winfried Boos,2 and Helena Santos1,*

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780 Oeiras, Portugal,1 and Department of Biology, University of Konstanz, D-78434 Konstanz, Germany2

Received 29 December 1998/Accepted 19 March 1999

Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus 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 four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-alpha -glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an alpha -glucosidase, a p-nitrophenyl-alpha -D-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. A Km of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5'-phosphate is conserved in the T. litoralis enzyme.

* Corresponding author. Mailing address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2780 Oeiras, Portugal. Phone: 35114426146. Fax: 35114428766. E-mail: santos{at}itqb.unl.pt.

Journal of Bacteriology, June 1999, p. 3358-3367, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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