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Originally published In Press as doi:10.1074/jbc.M404955200 on September 13, 2004

J. Biol. Chem., Vol. 279, Issue 46, 47890-47897, November 12, 2004
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TreT, a Novel Trehalose Glycosyltransferring Synthase of the Hyperthermophilic Archaeon Thermococcus litoralis*

Qiuhao Qu, Sung-Jae Lee, and Winfried Boos{ddagger}

From the Department of Biology, University of Konstanz, 78457 Konstanz, Germany

Received for publication, May 4, 2004 , and in revised form, August 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The gene cluster in Thermococcus litoralis encoding a multicomponent and binding protein-dependent ABC transporter for trehalose and maltose contains an open reading frame of unknown function. We cloned this gene (now called treT), expressed it in Escherichia coli, purified the encoded protein, and identified it as an enzyme forming trehalose and ADP from ADP-glucose and glucose. The enzyme can also use UDP- and GDP-glucose but with less efficiency. The reaction is reversible, and ADP-glucose plus glucose can also be formed from trehalose and ADP. The rate of reaction and the equilibrium favor the formation of trehalose. At 90 °C, the optimal temperature for the enzymatic reaction, the half-maximal concentration of ADP-glucose at saturating glucose concentrations is 1.14 mM and the Vmax is 160 units/mg protein. In the reverse reaction, the half-maximal concentration of trehalose at saturating ADP concentrations is 11.5 mM and the Vmax was estimated to be 17 units/mg protein. Under non-denaturating in vitro conditions the enzyme behaves as a dimer of identical subunits of 48 kDa. As the transporter encoded in the same gene cluster, TreT is induced by trehalose and maltose in the growth medium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trehalose synthesis in response to osmotic stress is observed in many organisms. For instance, in Escherichia coli trehalose is formed by the gene products of otsA and otsB catalyzing the transfer of glucose from UDP-glucose onto glucose-6-P (trehalose-6-P synthase), followed by the formation of trehalose (trehalose-6-P phosphatase) (1). This is the usual pathway for trehalose synthesis in most organisms. Another enzymatic reaction, catalyzed by the treS gene product, transforms maltose into trehalose in an equilibrium reaction (2). A third possibility is realized in some hyperthermophilic organisms. Here, the terminal {alpha} (1–4)-linked unit of a linear maltodextrin is converted into an {alpha},{alpha} (1–1) linkage by maltooligosyltrehalose synthase (encoded by treY). The terminal trehalose is then released by an additional enzyme, maltooligosyltrehalose trehalohydrolase (encoded by treZ) (3). Formally, trehalose phosphorylase (4) forming glucose and glucose-1-P from trehalose may also be regarded as a trehalose-synthesizing enzyme because the reaction is reversible at least in vitro. However, there is little doubt that the function of trehalose phosphorylase in vivo is in trehalose degradation rather than synthesis.

Trehalose metabolism, aside from the function of trehalose phosphorylase, is usually achieved by trehalase, an enzyme hydrolyzing trehalose to glucose (5–7). In Gram-negative enteric bacteria such as E. coli, degradation of trehalose is initiated by its uptake via enzyme IIBC of the phosphotransferase system under simultaneous phosphorylation to trehalose-6-P, followed by cytoplasmic hydrolysis of the latter to glucose and glucose-6-P mediated by trehalose-6-P hydrolase (8, 9).

The hyperthermophilic archaeon Thermococcus litoralis accumulates trehalose in response to high osmolarity when grown in the presence of yeast extract (10) that contains trehalose. Indeed, trehalose induces a high affinity and binding protein-dependent ABC transporter for trehalose and maltose that is most likely responsible for the accumulation of trehalose. This operon is also induced under conditions of elevated temperature (11). Thus, it is understood that the accumulation of trehalose occurs in response to osmotic, and possibly heat, stress. However, trehalose must also be metabolized in T. litoralis, albeit slowly because trehalose is used up in the stationary phase of growth after its accumulation. The enzymes for trehalose synthesis and degradation have not been identified in T. litoralis. Dialyzed cellular extracts obtained from cells grown in the presence of trehalose do not hydrolyze or otherwise modify trehalose.

In the past we studied the function of proteins encoded by a gene cluster in T. litoralis that appears in nearly identical sequence in Pyrococcus furiosus as the result of a lateral gene transfer (12). This cluster contains genes for a binding protein-dependent ABC transporter for trehalose and maltose (13–17) as well as a gene encoding the maltose-inducible repressor for the operon (18). The last unidentified gene in this cluster, now called treT (appearing in identical sequence in P. furiosus, PF 1742), shows homology to trehalose phosphorylases from fungi. Here we report that the enzyme exhibits a novel activity to reversibly transfer the glucose moiety of ADP-glucose onto glucose to form trehalose.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—[14C]trehalose (600–720 µCi/µmol) and purified E. coli trehalase were obtained from Trenzyme GmbH (www.trenzyme.com). All other commercial chemicals and enzymes used in this study were purchased from Sigma.

Cloning of treT, Overexpression and Purification of the Recombinant Protein—Two primers were designed based on the annotated sequence of the putative trehalose synthase (protein ID: AAG45375 [GenBank] PF1742). The forward primer was 5'-CGGGATCCATGTATGAGGTAACGAAGTTTGGTGGA-3' and the reverse primer was 5'-GCGTCGACAAAAGAATTTAGTAAATCAAGATACCTCTCAAG-3' with the BamHI and SalI restriction sites in bold, respectively. The chromosomal DNA was used as a template for PCR prepared as previously described (18). The PCR product was cloned into plasmid pET24a(+) (Novagen, Inc.) encoding a C-terminal His tag. The resulting plasmid was transformed into E. coli strain BL21. The transformed strain was grown at 28 °C in 4 liters of NZA medium (10 g NZ-amine A; Sheffield Product Inc.), 5 g of yeast extract, and 7.5 g of NaCl in 1 liter of distilled water) and was induced with 0.1 mM isopropyl-{beta}-D-thiogalactopyranoside overnight after the A578 of the culture had reached 1.0.

The cells were harvested by centrifugation and resuspended in 50 mM Tris-HCl, pH 7.5, containing 5 mM MgCl2 and 500 mM NaCl. The suspension was extracted by passing it three times through a French pressure cell at 16,000 p.s.i and 4 °C. The cell debris was removed by centrifugation at 19,000 x g for 10 min. The supernatant was heated to 80 °C for 10 min and centrifuged at 19,000 x g for 20 min.

The supernatant was loaded onto a nickel-nitrilotriacetic acid (Ni-NTA)1 affinity column equilibrated with 50 mM Tris-HCl, pH 7.5, as the first step of purification. The column was washed twice with the same Tris-HCl buffer containing 20 and 50 mM imidazole, respectively. Bound protein was subsequently eluted with 200 mM imidazole and passed through a desalting column (Econo-Pac 10 DG; Bio-Rad) to remove the imidazole. The preparation was purified further by molecular sieve chromatography on a Superdex 200 column (Amersham Biosciences) at 4 °C equilibrated with phosphate-buffered saline buffer containing 1 mM {beta}-mercaptoethanol and 5 mM MgCl2 to remove contaminants. Purified proteins were analyzed in 12% SDS-polyacrylamide gels and Western blots using anti-His tag antibodies from mouse (Qiagen).

Molecular Mass Determination—Gel filtration chromatography was performed on a Superdex 200 column (Amersham Biosciences) at 4 °C equilibrated with phosphate-buffered saline containing 1 mM {beta}-mercaptoethanol and 5 mM MgCl2. The low molecular mass calibration kit (Amersham Biosciences) was used consisting of bovine pancreas chymotrypsinogen A (25 kDa), hen egg albumin (43 kDa), bovine pancreas serum albumin (67 kDa), and rabbit muscle aldolase (158 kDa).

Thin Layer Chromatography (TLC)—Enzyme reaction mixtures were prepared in a total volume of 100 µl at 80 °C. In the trehalose synthesis (forward) reaction, 10 mM glucose, 10 mM ADP-glucose, 20 mM MgCl2, and 0.5 µg of enzyme were incubated in 50 mM Tris-HCl, pH 6.5. Samples were removed at different time intervals and 6 µl were spotted onto silica gel plates (type 60; Merck) that were then developed with butanol:ethanol:water (5:3:2) as solvent. Sugar-containing compounds were visualized by dipping the dried plate into methanol containing 5% H2SO4 followed by charring at 120 °C for 5 min.

Enzymatic trehalose degradation (reverse reaction) was done in 100 µl containing either 10 mM unlabeled trehalose or 47 µM [14C]trehalose and 10 mM ADP in the presence of 20 mM MgCl2 at 80 °C. Samples were removed at different time intervals and separated by TLC. The products formed were analyzed by charring or by autoradiography.

The control experiments were performed in the trehalose synthesis as well as the degradation reaction with either denatured enzyme or no enzyme in the mixtures described above. The enzyme was denatured by autoclaving at 130 °C for 30 min.

HPLC—Trehalose formation was also determined using HPLC (high pH-anion exchange chromatography) (Dionex Corp., Mississauga, Ontario) with pulsed amperometric detection. 100 µl of reaction mixture containing 8 mM ADP-glucose, 10 mM MgCl2, and varying glucose concentrations in 50 mM Tris-HCl, pH 6.5, were incubated at 90 °C. The reactions were started with the enzyme and stopped when still linear by adding cold buffer to a volume of 1 ml. The mixture was then kept on ice for 20 min. A Dionex carboPac MA-1 analytical column (4 x 250) equilibrated with 100 mM sodium acetate and 500 mM NaOH for the separation of monosaccharides and disaccharides was injected with 20 µl of reaction mixture. The quantities of glucose and trehalose were calculated by comparison with sugar standards (1 mM).

Kinetic Constants—Trehalose synthase activity (initial rates) was measured in both directions with discontinuous coupled enzyme assays. For trehalose synthesis, the half-maximal substrate concentration and Vmax for ADP-glucose was determined following ADP formation (Method 1). 100 µl of reaction mixture containing 20 mM glucose, 10 mM MgCl2, and varying ADP-glucose concentrations in 50 mM Tris-HCl, pH 6.5, were incubated for 6 min at 90 °C. The reaction was started by adding enzyme and stopped by adding cold buffer to a final volume of 980 µl. After cooling on ice for 20 min, the ADP produced in the reaction mixture was determined spectrophotometrically by adding 0.8 units of pyruvate kinase and 1.35 units of lactate dehydrogenase (Sigma), 0.3 mM NADH, and 2 mM phosphoenolpyruvate at 25 °C. The decrease in the absorption at 340 nm was followed in a total volume of 1 ml.

The half-maximal glucose concentration for ADP-glucose-dependent trehalose formation was determined using HPLC (see above) to follow the initial rate of trehalose formation. The reverse reaction (trehalose degradation) was measured by following the initial rate of glucose formation (Method 2). 100 µl of reaction mixture contained 5 mM ADP, 20 mM MgCl2, and varying trehalose concentrations in 50 mM Tris-HCl, pH 6.5, at 90 °C. The reaction was started by adding 0.5 µg of pure enzyme and stopped by adding 380 µl of cold buffer. Then the following coupled enzyme assay was performed at 25 °C: 0.5 units of hexokinase and glucose-6-P dehydrogenase, a final concentration of 0.2 mM ATP, 1 mM MgCl2 and 0.4 mM NADP+ were added to a final volume of 500 µl. The NADPH produced was measured at 340 nm.

For determining substrate specificity, instead of glucose, potential sugar acceptors were used in a concentration of 10 mM in the forward reaction. In the reverse reaction trehalose was replaced by potential disaccharide substrates at 10 mM.

All tests were done in duplicate, and all auxiliary enzymes were not rate-limiting. One unit of enzyme activity is defined as 1 µmol substrate formed/min at 90 °C. Specific enzyme activity is referred to as units/mg protein.

Induction by Different Sugars—T. litoralis was cultivated in Bacto Marine Broth medium (10) supplemented only with peptone (5 g/liter) as a control or with peptone plus maltose (3 g/liter), trehalose (3 g/liter), or sucrose (3 g/liter) for TreT induction. Cells were grown overnight at 80 °C, harvested by centrifugation (8,000 rpm, 4 °C), and extracted by sonication in an anaerobic chamber under a N2:H2 (95:5, v:v) atmosphere in 50 mM Tris-HCl, pH 7.5, containing 5 mM MgCl2 and 150 mM NaCl. Thin layer chromatography to measure trehalose formation was performed as described above. For Western blotting, the uninduced and induced cell extracts were loaded onto a 12% SDS-PAGE gel, transferred, blotted with a Fluorotrans transfer membrane (Pall Europe Limited), and incubated with anti-TreT antibodies raised in rabbit.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
treT Encodes an Enzyme Involved in Trehalose Metabolism as Indicated by Sequence Similarity—Fig. 1 shows the operon encoding the ABC transporter for trehalose and maltose (14) and the gene for an ATP-dependent fructokinase (frk) in T. litoralis (19). This gene cluster is contained in a 16-kb fragment of T. litoralis that appears to be the result of a horizontal gene transfer (of a composite transposon) between T. litoralis and P. furiosus (12). The operon encoding the ABC transporter is induced by maltose and trehalose with TrmB acting as a transcriptional repressor (18). From its encoded transport proteins and profile of induction by maltose and trehalose, one would assume that the role of the gene cluster is in sugar metabolism. In this respect it was of interest what treT, the only remaining gene of unidentified function, would encode. NCBI BLAST results revealed a conserved domain found in glycosyl transferases near the C terminus of TreT. The five best hits concern hypothetical proteins from other hyperthermophiles, for instance, PH1035 of Pyrococcus horikoshii (77% identity) or TM 0392 of Thermotoga maritima (53% identity). Also, there is high sequence similarity with two enzymes with trehalose phosphorylase activity from fungi, Pleurotus sajor-caju (31% identity) and Grifola frondosa (30% identity). Interestingly, TreT of T. litoralis also has low sequence similarity to a sucrose synthase from potato (EC 2.4.1.13 [EC] ; 25% identity). This enzyme performs the reversible transfer of glucose via UDP-glucose onto fructose to form sucrose. Fig. 2 shows a comparison of the deduced amino acid sequence of TreT with four other protein sequences, including a hypothetical protein from P. horikoshii, trehalose phosphorylase from fungi, and sucrose synthase from potato as given by Clustal W multiple sequence alignment.



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  		FIG. 1.
The T. litoralis ABC transporter gene cluster and the products of its genes. Arrows indicate the direction of transcription of the individual genes.

 



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  		FIG. 2.
Sequence comparison. Multiple alignment of the amino acid sequence of TreT with those of a hypothetical protein from P. horikoshii (PH1035), trehalose phosphorylase from P. sajor-caju (EC 2.4.1.64 [EC] ), trehalose synthase from G. frondosa (EC 2.4.1.64 [EC] ), and sucrose synthase from potato. Asterisks indicate identical amino acids; colons, high amino acid conservation; dots, low conservation in all five proteins as given by Clustal W.

 
Cloning and Heterologous Expression of treT and Purification of the Product—Amplified chromosomal treT DNA from T. litoralis was digested with two restriction endonucleases and cloned under isopropyl-{beta}-D-thiogalactopyranoside (IPTG) control into expression vector pET 24a(+) as a C-terminal His tag version. Fig. 3A shows an SDS-PAGE analysis of total cellular proteins with and without induction by IPTG. Cellular extracts of IPTG-induced cells were heated to 80 °C for 10 min prior to purification of the clarified extracts by Ni-NTA affinity chromatography. TreT is a protein of 413 amino acids with a calculated molecular mass of 48,146 Da. After the first purification step, two bands of ~48 and 37 kDa were seen in 12% SDS-polyacrylamide gels. Molecular sieve chromatography under non-denaturating conditions led to the purification of the larger protein exhibiting a molecular mass of 104,000 Da (Fig. 3C), indicating that TreT is a dimer of two identical polypeptides of 48 kDa.



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  		FIG. 3.
Purification and molecular mass determination of TreT. A, 12% SDS-PAGE of TreT. Lane 1, protein standards; lane 2, uninduced cell extract; lane 3, induced cell extract; lane 4, TreT after Ni-NTA affinity chromatography; lane 5, TreT further purified by molecular sieve chromatography on Superdex 200. B, Western blot analysis of TreT purification using anti-His tag antibodies. Lane 1, protein standards; lane 2, TreT after Ni-NTA affinity chromatography; lane 3, purified single band of TreT after molecular sieve chromatography; lane 4, uninduced cell extract; lane 5, induced cell extract. C, gel filtration chromatography of TreT on Superdex 200. Protein standards used: 1, rabbit muscle aldolase (158 kDa); 2, bovine pancreas serum albumin (67 kDa); 3, hen egg albumin (43 kDa); 4, bovine pancreas chymotrypsinogen A (25 kDa).

 
The two proteins of 48 and 37 kDa were eluted from the Ni-NTA column, and both carried the C-terminal His tag as seen in Western blots using anti-His tag antibodies (Fig. 3, A and B). The smaller protein most likely is not a degradation product of the larger polypeptide but the result of a restart within the treT open reading frame, as has also been found in the malK-encoded protein contained in the same operon (20). Purified TreT (48 kDa) is stable after heat treatment of the E. coli cellular extract. ~2 mg of pure protein were obtained routinely from 1 liter of E. coli culture.

TreT Is an ADP-glucose-dependent Trehalose Glycosyltransferring Synthase—We performed several tests with purified TreT to elucidate its enzymatic activity. We tested several disaccharides (maltose, sucrose, trehalose, lactose) and polysaccharides (linear maltodextrins, pullulan) and combinations of polysaccharides with disaccharides for their ability to act as substrates for a putative hydrolytic, phosphorolytic, or transfer activity of the enzyme. We also used glucose-6-P as acceptor for a possible glucosyltransfer from UDP-glucose or glucose-1-P to form trehalose-6-P or trehalose. None of these tests gave a positive result. However, when incubating ADP-glucose alone with the enzyme at 85 °C a product was slowly formed that could be identified by TLC as trehalose. ADP-glucose in the absence of the enzyme was then found to be unstable at this temperature and the buffer conditions used to slowly form free glucose. We then realized that the slow formation of trehalose was due to the transfer of glucose from ADP-glucose onto the glucose that had been liberated from ADP-glucose. Indeed, when glucose was included in the reaction mixture from the beginning, fast and time-dependent formation of trehalose could be observed (Fig. 4A). The identity of the formed product with trehalose was established by the action of E. coli periplasmic trehalase (6). This enzyme is specific for {alpha},{alpha} (1–1) glucosyl glucoside (trehalose) and does not hydrolyze {alpha}-or {beta}-paranitrophenyl glucosides or maltose, sucrose, lactose, or cellobiose (Fig. 4A).



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  		FIG. 4.
Identification of enzymatic products in the trehalose synthesis and degradation reactions by TLC. Samples of the incubation mixture were taken after different time intervals in the presence of enzyme. A, forward reaction in the presence of 10 mM glucose and 10 mM ADP-glucose. Lanes 1–6, 0, 5, 10, 15, 20, and 30 min of incubation; lane 7, sample from lane 6 treated with E. coli trehalase for 1 h at room temperature; lanes 8–12, sugar standards: glucose, trehalose, ADP-glucose, glucose-1-P, and glucose-6-P; lane 13, sugar standards, from top to bottom: glucose, trehalose, and ADP-glucose; lanes 14–23, cellobiose, maltose, sucrose, lactose, or trehalose incubated with E. coli trehalase. Samples were taken immediately after the addition of enzyme and after 1 h at room temperature each. B, forward reaction in the presence of [14C]glucose and 10 mM ADP-glucose. Lanes 1–8, reaction mixture incubated for 0, 3, 5, 10, 15, 20, 25, and 30 min; lane 9, [14C]glucose; lane 10, [14C]trehalose. C, reverse reaction using 10 mM trehalose and 10 mM ADP (lanes 1–5) or 50 mM phosphate (lanes 6–10) after incubation for 0, 5, 10, 20, and 40 min, respectively. Lanes 11–13, sugar standards: lane 11, (from top to bottom) glucose, trehalose, ADP-glucose; lane 12, glucose-1-P; lane 13: glucose-6-P. D, reverse reaction using [14C]trehalose and 10 mM ADP. Lane 1, [14C]glucose; lane 2, [14C]trehalose; lanes 3–10, reaction mixture incubated for 0, 1, 2, 3, 5, 10, 20, 40, and 60 min.

 
The trehalose-synthesizing reaction never went to completion, indicating that it is reversible. At high ADP-glucose concentration (10 mM) and trace amounts of [14C]glucose, the reaction went to near completion. This demonstrates its reversible nature. When starting the reaction with trehalose and ADP (i.e. the reverse reaction), we observed the formation of ADP-glucose and glucose (Fig. 4, C and D). Using [14C]trehalose the newly formed radioactive spot (aside from glucose) migrated identically and can be superimposed with chemically stained ADP-glucose (Fig. 4D, lane 11; charring is not shown). No enzymatic reaction can be detected when phosphate is used instead of ADP. Using autoclaved TreT as a control in both the trehalose synthesis and degradation reactions, no product formation can be detected (not shown). In the ADP-glucose-forming reaction, the amount of enzyme needed to obtain the same rate of reaction is ten times higher than for trehalose formation. Under these conditions, the reaction starts fast toward ADP-glucose, but the equilibrium is shifted strongly in the direction of trehalose formation. This can also be seen when the reaction is started with trace amounts of radiolabeled trehalose and 10 mM ADP (Fig. 4D). The formation of free glucose in this reaction is not caused by unspecific degradation of trehalose at the assay temperature because trehalose is stable under the conditions used (not shown). The stability of trehalose at high temperature had been demonstrated previously (21). Trehalose synthesis was also followed using HPLC. At varying initial glucose concentrations (3.5, 5, and 7.5 mM) but identical TreT concentrations, a new peak emerged that corresponded to standard trehalose (16.35-min retention time). The trehalose concentration obtained in this way after 7 min increased in response to the initial glucose concentration (Fig. 5).



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  		FIG. 5.
Identification of enzymatic products of the forward reaction by HPLC. The retention time of standard trehalose and glucose were 16.35 and 22.00 min, respectively. Three enzyme mixtures were prepared with increased glucose concentration at 3.5, 5.0, and 7.5 mM but constant ADP-glucose (8 mM) at 90 °C for 7 min. Three peaks appeared at different retention times: the injection peak, trehalose (16.35 min), and glucose (22.00 min).

 
Kinetic Constants, Temperature Dependence, and Substrate Specificity of TreT—The kinetics of trehalose glycosyltransferring synthase activity were measured in discontinuous two-step coupled assays. The half-maximal concentration of ADP-glucose at saturating glucose concentrations was 1.14 mM (Fig. 6A) and that for glucose at saturating ADP-glucose concentrations was 6.2 mM (Fig. 6B) as determined by HPLC. The Vmax for the purified enzyme was 160 units/mg protein for ADP-glucose at saturating glucose concentration and 220 units/mg protein for glucose at saturating ADP-glucose concentration. In the reverse reaction, the half-maximal concentration of trehalose was 11.5 mM, and the Vmax was estimated to be 17 units/mg protein (Fig. 6C). Thus, under saturating substrate concentrations, the enzymatic reaction is strongly favored toward the formation of trehalose.



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  		FIG. 6.
Kinetic analysis of the forward and the reverse reaction. The initial rate of trehalose formation was used to determine the concentration dependence of ADP-glucose (A) and glucose (B) in the forward reaction. C, in the reverse reaction, the formation of ADP was followed to determine the concentration dependence of trehalose. Values for Km and Vmax were extrapolated from the figures shown.

 
The effect of temperature (30–100 °C) on the enzymatic activity was measured with Method 1 at pH 6.5. The optimum for the trehalose-forming reaction was 90 °C (Fig. 7) with 65% enzymatic activity remaining at 100 °C. TreT showed a narrow pH optimum range around pH 6.5. At pH 7.5, only 13% of the activity at pH 6.5 remained.



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  		FIG. 7.
Effect of temperature on TreT activity.

 
Sugar specificity was tested in the directions of disaccharide synthesis as well as degradation. Fructose, mannose, galactose, xylose, 2-deoxy-glucose, glucosamine, {alpha}-methylglucoside, sorbitol, G-1-P, G-6-P, F-1-P, and F-6-P were unable to replace glucose as an acceptor for the glucosyl transfer from ADP-glucose when tested with Method 1. In addition, maltose, lactose, and sucrose were unable to be cleaved in the presence of ADP when tested with HPLC. Phosphate and P-enolpyruvate could not substitute for ADP in trehalose degradation as tested by using [14C]trehalose as substrate followed by TLC and autoradiography. Thus, TreT appears to be specific for the synthesis and the degradation of trehalose.

With UDP-glucose or GDP-glucose replacing ADP-glucose, only about 6 and 5% of TreT activity remained, whereas TDP-glucose was inactive. Method 1 was used to test the influence of cations (at 20 mM concentration). The enzyme needs Mg2+ for its activity (set as 100%), which can be replaced to some extent by CaCl2 (91%), MnCl2 (78%), CoCl2 (71.6%), and NiCl2 (57%). No enzymatic activity could be seen with ZnCl2.

TreT Is Induced by Maltose and Trehalose in the Growth Medium—We obtained cellular extracts from T. litoralis grown in peptone alone (as a control) and in peptone plus maltose, trehalose, or sucrose. The dialyzed extracts at identical protein concentrations were tested for their ability to form trehalose from ADP-glucose and glucose. Trehalose formation was monitored by TLC and is shown in Fig. 8. When grown only on peptone, trehalose formation was hardly detectable (Fig. 8A). Induction was highest with maltose in the growth medium (Fig. 8B), less efficient with trehalose, and even less with sucrose, although with the latter it was clearly higher than in the extract of uninduced cells (grown on peptone alone). The induction of TreT by maltose and trehalose in the growth medium could also be detected in Western blots with anti-TreT antibodies from rabbit (not shown). The induction pattern of treT was identical to that of malE, one of the genes encoding the ABC transporter for trehalose and maltose, consistent with the notion that these genes form an operon together with treT (20, 22).



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  		FIG. 8.
TLC analysis of TreT induction by different sugars. A, trehalose formation by extracts of T. litoralis grown on peptone (lanes 2–6) and peptone plus sucrose (lanes 7–11) after 0, 5, 10, 20, and 40 min of incubation, respectively. B, trehalose formation by extracts of trehalose-induced (lanes 2–6) or maltose-induced cells (lanes 7–11) after 0, 3, 5, 7, and 10 min of incubation. A and B, lane 1, sugar standards: glucose, trehalose, ADP-glucose.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We describe an enzyme, TreT, from the hyperthermophilic archaeon T. litoralis that produces trehalose by the transfer of glucose from ADP-glucose (or, less effectively, UDP-glucose and GDP-glucose) onto the 1-position of glucose to form trehalose and ADP. The trehalose produced is identical with {alpha},{alpha}-trehalose because E. coli trehalase was able to hydrolyze it to glucose. Such an enzymatic activity for the synthesis of trehalose has not yet been described for any organism. Instead, when activated glucose in the form of UDP-glucose is used in the synthesis of trehalose, the transfer of the glucosyl moiety always occurs onto glucose-6-P to form trehalose-6-P, followed by enzymatic hydrolysis of the latter to trehalose (for instance, by the enzymes encoded by the E. coli otsA otsB genes). In this way, unidirectional formation of trehalose is assured. The trehalose-synthesizing reaction catalyzed by TreT is reversible. Thus, ADP-glucose and glucose can be formed from ADP and trehalose.

What is the physiological role of TreT? From the position of treT within a gene cluster encoding an ABC transporter for trehalose (and maltose), one would be inclined to interpret the function of TreT as a trehalose-degrading enzyme rather than a trehalose-synthesizing enzyme. This also appears sensible in light of the rather high half-maximal concentration for glucose of 6.2 mM. It would be unlikely that growing T. litoralis contains such high internal glucose concentrations. With a Km of 0.7 mM for glucose, the ADP-dependent hexokinase (22) would effectively reduce the concentration of free internal glucose and prevent any noticeable trehalose formation by TreT. One could argue that the metabolism of maltose, which also induces TreT synthesis, would provide ample amounts of free internal glucose (23) needed for trehalose formation by TreT. However, trehalose formation was not observed when T. litoralis grew on maltose (10). The accumulation of trehalose present in the medium via the high affinity and binding protein-dependent ABC transporter results in high internal trehalose concentrations (10) that could subsequently be used for glucose formation by TreT, followed by hexokinase-dependent phosphorylation and glycolysis (22). The fact that the formation of ADP-glucose is a necessary by-product of this pathway opens the possibility that it may be used for glucose polymer formation in the form of glycogen. Indeed, the major pathway of cellulose biosynthesis in plants and cyanobacteria follows this strategy (24). There, sucrose is used by an enzyme that forms UDP-glucose from UDP and sucrose. UDP-glucose is then used as a donor for cellulose biosynthesis (25). There is only residual sequence similarity of these types of enzymes with TreT described here. In contrast, significant sequence identity is seen with enzymes that are often described as putative trehalose synthases, and a few are characterized as authentic trehalose phosphorylases. These enzymes reversibly cleave trehalose by phosphorolysis to form glucose-{beta}-1-P and glucose (26). They usually function as trehalose-degrading enzymes rather than trehalose-synthesizing enzymes. The high similarity of these enzymes to TreT is intriguing. One is inclined to describe the ADP-glucose formation by TreT also as a phosphorolysis in which not free Pi but the {beta}-phosphoryl group of ADP is the active agent hydrolyzing trehalose.


   FOOTNOTES
 
* This work was supported by grants from the Deutsche Forschungs-gemeinschaft and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 0049-7531-882658; Fax: 0049-7531-883356; E-mail: winfried.boos{at}uni-konstanz.de.

1 The abbreviations used are: Ni-NTA, nickel-nitrilotriacetic acid; HPLC, high pH-anion exchange chromatography. Back


   ACKNOWLEDGMENTS
 
We acknowledge the contribution of Peter Kroth who made us aware of the similarity of the TreT function to the plant enzymes forming UDP-glucose from sucrose in cellulose biosynthesis. We thank Reinhold Horlacher for providing [14C]trehalose and purified trehalase, Xiangzhen Li for preparing T. litoralis cell extract, Christoph Mayer for measuring sugars with HPLC, Irena Hendekovic for kinetic analyses, and E. Oberer-Bley for critically reading the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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