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

Abstract

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 degrees C), of 66 U/mg. A Km 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 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.

FIG. 1

TLC of maltose reaction products obtained by a dialyzed T. litoralis cell extract (A), 4-α-glucanotransferase-free cell extract (B), and purified 4-α-glucanotransferase (C). Dialyzed cell extract (1.4 mg of protein per ml), glucanotransferase-free cell extract (4.6 mg of protein per ml), or purified glucanotransferase (11 μg of protein per ml) was incubated with 10 mM maltose in 100 mM MOPS (pH 7.0) at 85°C. Aliquots of the reaction mixture (10 μl) were applied to the TLC plate at different time intervals. S, standards; G₁, glucose; G₂, maltose; G₃, maltotriose; G₄, maltotetraose.

FIG. 2

SDS-PAGE of purified T. litoralis 4-α-glucanotransferase and maltodextrin phosphorylase. Lane 1, molecular mass standards; lane 2, purified glucanotransferase (Gtase; 79 kDa); lane 3, purified MalP (94 kDa).

FIG. 3

Temperature dependence of T. litoralis 4-α-glucanotransferase activity. The assay mixture, containing 7 μg of enzyme per ml in 100 mM MOPS (pH 7.0)–10 mM maltotriose, was incubated at different time points. Enzyme activity was assessed by the production of glucose as described in Materials and Methods.

FIG. 4

TLC of ¹⁴C-labeled reaction products of purified 4-α-glucanotransferase with unlabeled maltose and [U-¹⁴C]glucose (A) or with unlabeled maltose and [U-¹⁴C]maltose (B). The reaction mixture (45 μl) contained 1.6 μg of enzyme, 10 mM unlabeled maltose in 100 mM MOPS (pH 7.0), and 0.33 nmol of [U-¹⁴C]glucose or 0.6 nmol of [U-¹⁴C]maltose. Aliquots of the reaction mixture (10 μl) were applied to the TLC plate at different time intervals. G₁, glucose; G₂, maltose; G₃, maltotriose; G₄, maltotetraose.

FIG. 5

Temperature dependence of T. litoralis MalP activity. The assay mixture containing 2 μg of enzyme per ml in 50 mM potassium phosphate (pH 7.0)–5 mM maltoheptaose was incubated for 15 min. The enzyme activity was assessed by the production of glucose-1-phosphate as described in Materials and Methods. For all temperatures tested, glucose-1-phosphate production was linear with time.

FIG. 6

Sequence alignment of the MalP from T. litoralis (Tli MalP) with phosphorylases from other thermophilic organisms. Pho PH1512, homologous sequence from hyperthermophilic archaeon P. horikoshii OT3 (16); Tma AgpA, MalP from the hyperthermophilic bacterium T. maritima (1). The pyridoxal 5′-phosphate binding consensus sequence begins at position 584 of the Tli MalP sequence.

FIG. 7

TLC of maltotetraose reaction products obtained with T. litoralis cell extracts in the presence and absence of phosphate. Reaction mixtures containing 5 mM maltotetraose and the cell extract (1 mg of protein per ml) in 100 mM MOPS (pH 7.0) (A) or in 50 mM potassium phosphate (pH 7.0) (B) were incubated at 85°C. Aliquots of the reaction mixture (10 μl) were applied to the TLC plate at different time intervals. S, standards; G₁, glucose; G₂, maltose; G₃, maltotriose; G₄, maltotetraose; G-1-P, glucose-1-phosphate.

FIG. 8

TLC of maltose reaction products obtained with T. litoralis and P. furiosus cell extracts. Reaction mixtures of T. litoralis (A) and P. furiosus (B) dialyzed cell extracts (5 mg of protein per ml) were incubated with 10 mM maltose in 100 mM MOPS (pH 7.0) at 85 and 95°C, respectively. Aliquots of the reaction mixture (10 μl) were applied to the TLC plate at different time intervals. Notation is as in Fig. 1.

FIG. 9

Proposed pathway for maltose metabolism in T. litoralis. MalE, maltose/trehalose binding protein; MalF, MalG, and MalK, components of the maltose/trehalose ABC transport system; PGM, phosphoglucomutase; HK, hexokinase; PFK, phosphofructokinase; GAP:FdOR, glyceraldehyde-3-phosphate:ferredoxin oxidoreductase; Fd, ferredoxin; F-6-P, fructose-6-P; F-1,6-bP, fructose-1,6-bisphosphate.