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Trehalose (α,α-1,1-glucosyl-glucose) is essential for the growth of mycobacteria, and these organisms have three different pathways that can produce trehalose. One pathway involves the enzyme described in the present study, trehalose synthase (TreS), which interconverts trehalose and maltose. We show that TreS from Mycobacterium smegmatis, as well as recombinant TreS produced in Escherichia coli, has amylase activity in addition to the maltose trehalose interconverting activity (referred to as MTase). Both activities were present in the enzyme purified to apparent homogeneity from extracts of Mycobacterium smegmatis, and also in the recombinant enzyme produced in E. coli from either the M. smegmatis or the Mycobacterium tuberculosis gene. Furthermore, when either purified or recombinant TreS was chromatographed on a Sephacryl S-200 column, both MTase and amylase activities were present in the same fractions across the peak, and the ratio of these two activities remained constant in these fractions. In addition, crystals of TreS also contained both amylase and MTase activities. TreS produced both radioactive maltose and radioactive trehalose when incubated with [3H]glycogen, and also converted maltooligosaccharides, such as maltoheptaose, to both maltose and trehalose. The amylase activity was stimulated by addition of Ca2+, but this cation inhibited the MTase activity. In addition, MTase activity, but not amylase activity, was strongly inhibited, and in a competitive manner, by validoxylamine. On the other hand, amylase, but not MTase activity, was inhibited by the known transition-state amylase inhibitor, acarbose, suggesting the possibility of two different active sites. Our data suggest that TreS represents another pathway for the production of trehalose from glycogen, involving maltose as an intermediate. In addition, the wild-type organism or mutants blocked in other trehalose biosynthetic pathways, but still having active TreS, accumulate 10- to 20-fold more glycogen when grown in high concentrations (≥ 2% or more) of trehalose, but not in glucose or other sugars. Furthermore, trehalose mutants that are missing TreS do not accumulate glycogen in high concentrations of trehalose or other sugars. These data indicate that trehalose and TreS are both involved in the production of glycogen, and that the metabolism of trehalose and glycogen is interconnected.
Trehalose is a nonreducing disaccharide of d-glucose in which the two glucoses are linked in an α,α-1,1-glycosidic linkage [1,2]. Trehalose can play a number of different roles in biological systems, including serving as a reservoir of glucose for energy and/or carbon ; functioning as a stabilizer or protectant of proteins and membranes during times of stress ; acting as a regulatory molecule in the control of glucose metabolism ; serving as a transcriptional regulator ; and playing a structural and functional role as a component of various cell wall glycolipids in mycobacteria and related organisms .
In Mycobacterium smegmatis and related organisms, there are at least three different pathways that can give rise to trehalose [1,8]. The best known and most widespread pathway in many biological systems is referred to as the TPS/TPP or OtsA/OtsB pathway, which involves two enzymes. The first enzyme, trehalose phosphate synthase (TPS or OtsA), transfers glucose from UDP-glucose to glucose 6-phosphate to form trehalose phosphate and UDP . The second enzyme is a highly specific phosphatase, trehalose-phosphate phosphatase (TPP or OtsB), that removes the phosphate to produce free trehalose plus inorganic phosphate . A second pathway of more limited scope in biological systems also involves two enzymes that convert glycogen to trehalose . The first enzyme of this pathway is maltooligosyl trehalose synthase (TreY), which changes the α1-4 linkage at the reducing end of bacterial glycogen to the α,α,1,1-linkage of trehalose. The second enzyme, maltooligosyl trehalose trehalohydrolase (TreZ), cleaves the α1,4-glycosidic linkage to which the newly-formed trehalose is attached, producing free trehalose and leaving a glycogen chain minus two glucoses . The third pathway involves a single enzyme, trehalose synthase (TreS), which catalyzes the interconversion of maltose and trehalose [13,14]. Although TreS can produce trehalose from maltose, it has been postulated that its real role, at least in corynebacteria, is to control intracellular levels of trehalose by converting excess trehalose to maltose, which can then be converted by α-glucosidases to glucose [15,16]. By contrast, mycobacteria have a potent trehalase , whereas corynebacteria do not. Therefore, the TreS of mycobacteria may have a different and more significant role in the synthesis of trehalose from maltose. However, until now, it has not been clear where mycobacteria could obtain the maltose to transform into trehalose because M. smegmatis grows very poorly on maltose.
Our preliminary experiments suggested that TreS was somehow involved in glycogen synthesis and degradation. Thus, it was important to determine how the presence of TreS affects the levels of glycogen and trehalose in cells. Accordingly, mutants of M. smegmatis that were missing TreS or one of the other trehalose biosynthetic pathways were prepared (for designation of mutants, see Table 1) and the levels of glycogen and trehalose were compared in these cells. In addition, either recombinant TreS made in Escherichia coli, or TreS purified from the wild-type M. smegmatis, was assayed to determine its substrate specificity, and its sensitivity to various inhibitors of trehalose or glycogen metabolism. These studies demonstrated that TreS contains amylase activity, in addition to its maltose trehalose interconverting activity (referred to as MTase). These experiments also show that all of the M. smegmatis stains that contain TreS accumulate large amounts of glycogen when grown in high concentrations of trehalose, but mutants missing TreS activity do not accumulate glycogen, regardless of the amount of trehalose in the media. The results obtained indicate that TreS plays an key role in the utilization of trehalose for the production of glycogen. We hypothesize that TreS acts as a sensor or regulator of trehalose levels in these cells by catalyzing the conversion of glycogen to trehalose when cytoplasmic trehalose levels are low, but this enzyme also can expedite or promote the conversion of trehalose to glycogen when cytoplasmic trehalose levels become too high.
Table 1. Enzymatic profiles of various mycobacterial trehalose biosynthetic mutants.
|Mutant designation||Enzyme(s) missing (trehalose biosynthesis)||Trehalose biosynthetic pathways (active)|
|Wild-type||None||All (i.e. TPS/TPP; TreS TreY/TreZ)|
|#74||TPS, TPP, TreY||TreS|
|#80||TPS/TPP, TreS, TreY||None|
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- Experimental procedures
TreS is a 68 kDa protein that is present in a number of bacteria, including mycobacteria, corynebacteria, nocardia and streptomyces, as well as arthrobacter, sulfolobus and rhizobium [8,11–13]. TreS has been purified to near homogeneity from M. smegmatis, and the gene for this protein was cloned and expressed in E. coli . The expressed protein had a subunit molecular mass of 68 kDa on SDS gels, but active enzyme eluted as a 390 kDa protein upon gel filtration, suggesting that active TreS is a hexamer of six identical subunits. TreS catalyzes the reversible interconversion of trehalose and maltose. The reaction kinetics favor the conversion of maltose to trehalose, with a Km for maltose of approximately 10 mm, whereas the Km for trehalose is approximately 90 mm.
In Corynebacterium glutamicum, TreS has been proposed to function as a substitute for a trehalase to control intracellular levels of trehalose because no ORF homologous to known trehalase genes have been identified, nor has any trehalase activity been demonstrated in this organism . However, M. smegmatis does have a highly specific and active trehalase , in addition to the TreS described above . Another report on the TreS of C. glutamicum suggests that this enzyme is only involved in trehalose biosynthesis when these organisms are growing on maltose. Thus, a critical question with regard to the production of trehalose by TreS remains. What is the possible source of maltose that TreS could use as a substrate to produce trehalose?
Exogenous maltose is not a likely source of maltose for M. smegmatis because this organism grows very poorly on maltose. However, the results obtained in the present study indicate that endogenous maltose can be produced from glycogen by the amylase activity of TreS, and that this maltose is readily converted to trehalose by the MTase activity of TreS.
The present study provides evidence indicating that both activities reside in the same protein. First, TreS, purified from M. smegmatis as well as recombinant TreS produced in E. coli, had both MTase activity and amylase activity. Second, the 68 kDa TreS undergoes auto-proteolysis to give a 58 kDa protein, which also contains both MTase and amylase activity. Third, the 58 kDa protein was subjected to gel filtration and fractions were collected. Six fractions across the protein peak had variable amounts of MTase activity with the highest activity corresponding to fractions showing the most 58 kDa protein by SDS/PAGE. Importantly, the ratio of MTase/amylase, but not the absolute activity, remained fairly constant in fractions having different amounts of the 58 kDa protein. Finally, crystals of TreS were obtained, and these isolated crystals have both amylase activity and MTase activity.
These results strongly indicate that the MTase activity and the amylase activity are in the same protein, and suggest that this multifunctional protein has the capacity to convert glycogen to trehalose.
The partial amino acid sequence of TreS from M. smegmatis allowed us to locate the ORF for this protein and a blastp search indicated that it had approximately 83% identity to a gene (Rv 0126) for a hypothetical α-amylase in the M. tuberculosis genome . It also has 72% identity to a putative TreS from Streptomyces avermitilis, 69% identity in C. glutamicum, and 61% identity to the putative TreS from Pseudomonas sp. Because there are no reports on the isolation or characterization of these TreS proteins, it is not known whether they also have amylase activity, but it will be interesting to determine whether the TreS of corynebacteria also shares this activity. It will also be important to determine ways to test this amylase activity for function in vivo to establish whether it can really act in collaboration with the TreS activity to convert glycogen glucoses into cytoplasmic trehalose.
We propose that TreS has two distinct active sites: one catalyzing the interconversion of maltose and trehalose (referred to here as MTase activity) and the other catalyzing the breakdown of glycogen to maltose (amylase activity).
The present study provides evidence supporting the existence of the two sites. First, the amylase site is activated by Ca2+ whereas the MTase activity is inhibited by Ca2+ and other cations. Second, we have identified two inhibitors each of which competitively inhibits one activity and not the other. Thus, validoxylamine competitively inhibits MTase but not amylase, whereas acarbose competitively inhibits amylase but not MTase. Third, glycogen, which is a substrate for the amylase activity of TreS, has no effect on the MTase activity of TreS. That is, incubations of MTase with trehalose produce the same amount of maltose, even in the presence of high amounts of glycogen.
These data suggest that these two activities reside in different sites on the protein. However, it will require site-directed mutagenesis studies, or deletions of various parts of the protein, to conclusively prove that there are indeed two sites. Once we have identified active site amino acids for each catalytic activity, it will be possible to perform site-directed mutagenesis to modify one activity and not the other. We have been able to obtain small-sized crystals of TreS but they do not have sufficiently high resolution for structural analysis. Attempts to improve the resolution of these crystals is in progress.
Our hypothesis on the function of TreS is that it serves as a sensor and/or controller of the cellular trehalose levels in mycobacteria and perhaps other organisms. The present studies show that TreS can mediate the formation of trehalose from glycogen. In addition, growth studies with the wild-type M. smegmatis show that, when this organism is grown in a mineral salts medium with high concentrations (1–4%) of trehalose as the major carbon source, these cells contain 10- to 30-fold higher amounts of glycogen than cells grown in the same concentration of glucose or other sugars. Furthermore, additional studies with a number of trehalose mutants that are missing one, two or all three of the trehalose biosynthetic pathways (Table 1) demonstrate that any of the mutants still containing TreS (including the mutant that only has TreS) show this accumulation of glycogen in the presence of high trehalose, but any mutants that are missing TreS do not accumulate glycogen at any level of trehalose, or any other sugar. Thus, TreS not only is involved in the production of trehalose from glycogen, but also appears to play an essential role in the formation, and/or accumulation, of glycogen. This accumulation somehow involves the utilization of trehalose as the carbon source, but the mechanism of this conversion is not known. We propose that when high levels of trehalose are produced in the cell, perhaps as a result of exposure to stress, TreS may function to convert this trehalose to maltose and then to glycogen when the stress is removed. Removal of trehalose is probably essential because high levels of trehalose may be toxic. On the other hand, if trehalose falls to a dangerously low level, TreS may function to convert glycogen to maltose and then to trehalose. Ongoing studies are attempting to determine how trehalose is involved in the formation of glycogen, and how TreS functions as a sensor or regulator of trehalose and/or glycogen levels in these cells.