Unusual starch degradation pathway via cyclodextrins in the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324

Abstract

The hyperthermophilic archaeon Archaeoglobus fulgidus strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, alpha-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that A. fulgidus utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly beta-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from Thermococcus litoralis. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.

FIG. 1.

Growth of A. fulgidus strain 7324 on β-cyclodextrins at 76°C on medium containing 2 g of cyclodextrin liter⁻¹, 0.5 g of yeast extract liter⁻¹, and 30 mM sulfate. •, Cell number per ml; ▪, cyclodextrin concentration.

FIG. 2.

Purification and activity of CGTase from A. fulgidus strain 7324. (A) SDS-PAGE, with silver staining of partially purified CGTase. Lanes: 1, standard; 2, 1 μg of protein after Resource Phe. (B) Zymogram of CGTase from A. fulgidus strain 7324. Partially purified protein was used to identify the band containing CGTase activity. Lanes: 1, standard; 2, 10 μg of protein after the addition of Superdex 200 stained with Coomassie brilliant blue; 3, 10 μg of protein after the addition of Superdex 200 with activity staining.

FIG. 3.

Purification of CDase, Mal-P, and PGM from A. fulgidus strain 7324 by SDS-PAGE, with silver staining. Lanes: 1, standard; CDase, 0.3 μg of protein after Bioprep SE 1000/17; Mal-P, 0.2 μg of protein after Bioprep SE 1000/17; PGM, 0.3 μg of protein after UNO Q1.

FIG. 4.

Determination of reaction products of CDase from the substrates α-, β-, and γ-cyclodextrin as analyzed by TLC on silica gel 60. The assay contained 0.5% of the respective cyclodextrin and 2 μg of protein (+) in 50 mM sodium acetate (pH 4.5) incubated at 80°C for 1.5 h. −, Control assays without protein. The sugars were stained with 5% H₂SO₄ after 15 min of baking at 120°C. Lanes 1 and 2, α-cyclodextrin; lanes 3 and 4, β-cyclodextrin; lanes 5 and 6, γ-cyclodextrin; lane 7, standard for glucose units. Note that cyclodextrins showed smaller migration distances than the respective maltooligosaccharides, which corresponded to the glucose unit standard.

FIG. 5.

Substrate spectrum of Mal-P from A. fulgidus strain 7324. The relative activity of 100% corresponds to 34 U mg⁻¹. M4, maltotetraose; M5, maltopentaose; M6, maltohexaose; M7, maltoheptaose. The standard deviation was <5%.

FIG. 6.

Rate dependence of Mal-P from A. fulgidus strain 7324 on the concentration of maltoheptaose (A) and phosphate (B). The inset graphs show double reciprocal plots of the rates versus the corresponding substrate concentrations.

FIG. 7.

Proposed pathway of starch degradation to glucose-6-phosphate in the hyperthermophilic sulfate-reducing archaeon A. fulgidus strain 7324. The ABC transporter was postulated in analogy to K. oxytoca and Thermococcus sp. strain B1001 (15, 20). G-1-P, glucose-1-phosphate; G-6-P, glucose-6-phosphate. The formation of glucose by CGTase is not shown. Glucose and glucose-6-phosphate are degraded via a modified Embden-Meyerhof pathway to pyruvate.

FIG. 8.

Alignment of deduced amino acid sequence of CGTase from A. fulgidus strain 7324 (Af) and archaeal (cyclodextrin)GTases. The dots represent the substrate binding sites of the GH 57 family, the squares show the proposed calcium binding sites, and the arrowheads indicate the conserved catalytic residues of the GH 57 family. The four conserved regions typical for enzymes of the GH 13 family are indicated by boxes and designated I, II, III, and IV. The invariant amino acids of the GH 13 family within the conserved regions are marked by asterisks. The alignment was constructed by using CLUSTAL X (68). Identical residues are indicated by dark shading; conserved substitutions are indicated by light shadings. NCBI accession numbers: Tl, T. litoralis D88253; Tk, T. kodakaraensis (AB072372); Pf, P. furiosus ABA33720.1; Tsp, Thermococcus sp. strain B1001 BAA88217.1.

FIG. 9.

Phylogenetic relationship of CGTase from A. fulgidus strain 7324 (AfCGT) with members of the GH 13 and GH 57 families. The tree was constructed by the neighbor-joining method of CLUSTAL X (68). The given numbers represent bootstrapping values according to the neighbor-joining method. NCBI accession numbers: T. litoralis (TlitGT), D88253; Thermococcus sp. strain B1001 (TspCGT), BAA88217.1; T. kodakaraensis (TkodCGT), BAB78538.1; P. furiosus CGTase (PfuCGT), ABA33720.1; A. gottschalkii (AgotCGT), CAH61550.1; K. oxytoca (KoxyCGT), P08704; Geobacillus stearothermophilus (BsteCGT), CAA41770.1; Bacillus macerans (BmacCGT), P04830; P. furiosus alpha-amylase (PfuGH57), AAA72035.1; T. kodakaraensis 4-α-glucanotransferase (TkodGH57), BAA22062.1.