We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 M and 0.5 mM sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80°C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

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 halfmaximal concentration of ADP-glucose at saturating glucose concentrations is 1.14 mM and the V max 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 V max 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.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 ␣ (1-4)-linked unit of a linear maltodextrin is converted into an ␣,␣ (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 (...

Background: The efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI) in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII) in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS), which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS.

BackgroundIn microorganisms and plants, the first two reactions of arginine biosynthesis are catalyzed by N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK). In mammals, NAGS produces an essential activator of carbamylphosphate synthetase I, the first enzyme of the urea cycle, and no functional NAGK homolog has been found. Unlike the other urea cycle enzymes, whose bacterial counterparts could be readily identified by their sequence conservation with arginine biosynthetic enzymes, mammalian NAGS gene was very divergent, making it the last urea cycle gene to be discovered. Limited sequence similarity between E. coli NAGS and fungal NAGK suggests that bacterial and eukaryotic NAGS, and fungal NAGK arose from the fusion of genes encoding an ancestral NAGK (argB) and an acetyltransferase. However, mammalian NAGS no longer retains any NAGK catalytic activity.ResultsWe identified a novel bifunctional N-acetylglutamate synthase and kinase (NAGS-K) in the Xanthomonadales order of gamma-proteobacteria that appears to resemble this postulated primordial fusion protein. Phylogenetic analysis indicated that xanthomonad NAGS-K is more closely related to mammalian NAGS than to other bacterial NAGS. We cloned the NAGS-K gene from Xanthomonas campestis, and characterized the recombinant NAGS-K protein. Mammalian NAGS and its bacterial homolog have similar affinities for substrates acetyl coenzyme A and glutamate as well as for their allosteric regulator arginine.ConclusionThe close phylogenetic relationship and similar biochemical properties of xanthomonad NAGS-K and mammalian NAGS suggest that we have identified a close relative to the bacterial antecedent of mammalian NAGS and that the enzyme from X. campestris could become a good model for mammalian NAGS in structural, biochemical and biophysical studies.