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Journal of Bacteriology, June 1999, p. 3358-3367, Vol. 181, No. 11
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-
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
-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--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 -glucosidase, a
p-nitrophenyl--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.
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