The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A.

We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis. In vivo, this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli. It consists of two similar lobes, each formed by a parallel beta-sheet flanked by alpha-helices on both sides. Both are connected by a hinge region consisting of two antiparallel beta-strands and an alpha-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

Evidence of recent lateral gene transfer among hyperthermophilic archaea.

A total of 153 nucleotide differences were found over a contiguous 16 kb region between two hyperthermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis. The 16 kb region in P. furiosus is flanked by insertion sequence (IS) elements with inverted and direct repeats. Both IS elements contain a single open reading frame (ORF) encoding a putative protein of 233 amino acids identified as a transposase. This 16 kb region has the features of a typical bacterial composite transposon and represents a possible mechanism for lateral gene transfer between Archaea or possibly between Archaea and Bacteria. A total of 23 homologous IS elements was found in the genome sequence of P. furiosus, whereas no full-length IS elements were identified in the genomes of Pyrococcus abyssi and Pyrococcus horikoshii. Only one IS element was found in T. litoralis. In P. furiosus and T. litoralis, the 16 kb region contains an ABC transport system for maltose and trehalose that was characterized biochemically for T. litoralis. Regulation of expression studies showed that the malE gene, located on the transposon, and the encoded trehalose/maltose-binding protein (TMBP) are induced in the presence of maltose and trehalose in both P. furiosus and T. litoralis. The implications of transposition as a mechanism for lateral gene transfer among Archaea are discussed.

Molecular and biochemical analysis of MalK, the ATP-hydrolyzing subunit of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis.

We report the cloning, sequencing, and expression of malK encoding the ATP-hydrolyzing subunit of the maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis. According to the deduced amino acid sequence, MalK consists of 372 amino acids with a calculated molecular weight of 41,787. It shows 47% identity with the MalK protein of Escherichia coli and high sequence conservation in important regions. C-terminal His-tagged MalK was purified. The soluble protein appeared monomeric by molecular sieve chromatography and showed ATPase activity. Enzymatic activity was highest at 80 degrees C with a Km of 150 microM and a Vmax of 0.55 micromol of ATP hydrolyzed/min/mg of protein. ADP was not a substrate but a competitive inhibitor (Ki 230 microM). GTP and CTP were also hydrolyzed. ATPase activity was inhibited by N-ethylmaleimide but not by vanadate. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the ABC transporters in these two phylogenetic branches.

Crystal structure of the effector-binding domain of the trehalose-repressor of Escherichia coli, a member of the LacI family, in its complexes with inducer trehalose-6-phosphate and noninducer trehalose.

The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose-6-phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 A resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 A resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central beta-sheet sandwiched between alpha-helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose-6-phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose-6-phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source.

The periplasmic cyclodextrin binding protein CymE from Klebsiella oxytoca and its role in maltodextrin and cyclodextrin transport.

Klebsiella oxytoca M5a1 has the capacity to transport and to metabolize alpha-, beta- and gamma-cyclodextrins. Cyclodextrin transport is mediated by the products of the cymE, cymF, cymG, cymD, and cymA genes, which are functionally homologous to the malE, malF, malG, malK, and lamB gene products of Escherichia coli. CymE, which is the periplasmic binding protein, has been overproduced and purified. By substrate-induced fluorescence quenching, the binding of ligands was analyzed. CymE bound alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin, with dissociation constants (Kd) of 0.02, 0.14 and 0.30 microM, respectively, and linear maltoheptaose, with a Kd of 70 microM. In transport experiments, alpha-cyclodextrin was taken up by the cym system of K. oxytoca three to five times less efficiently than maltohexaose by the E. coli maltose system. Besides alpha-cyclodextrin, maltohexaose was also taken up by the K. oxytoca cym system, but because of the inability of maltodextrins to induce the cym system, growth of E. coli mal mutants on linear maltodextrin was not observed when the cells harbored only the cym uptake system. Strains which gained this capacity by mutation could easily be selected, however.

Archaeal binding protein-dependent ABC transporter: molecular and biochemical analysis of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis.

We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85 degrees C with a Kd of 0.16 microM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

Characterization of TreR, the major regulator of the Escherichia coli trehalose system.

The pathway of trehalose utilization in Escherichia coli is different at low and high osmolarity. The low osmolarity system takes up trehalose as trehalose 6-phosphate which is hydrolyzed to glucose and glucose 6-phosphate. treB and treC, the genes for the enzymes involved, form an operon that is controlled by TreR (encoded by treR), the repressor of the system, for which trehalose 6-phosphate is the inducer. We have cloned and sequenced treR. The protein contains 315 amino acids with a molecular weight of 34,508. TreR was purified and shown to bind as a dimer trehalose 6-phosphate and trehalose with a Kd of 10 and 280 microM, respectively. The conformations of the protein differ from each other with either one or the other substrate-bound. Protease treatment removed the DNA-binding domain from the intact protein leaving the dimerization domain (a 29-kDa carboxyl-terminal fragment) intact. Nuclease protection experiments revealed a palindromic sequence located directly upstream of the -35 promoter sequence of treB that functions as the operator of the system.

Molecular characterization of glucokinase from Escherichia coli K-12.

glk, the structural gene for glucokinase of Escherichia coli, was cloned and sequenced. Overexpression of glk resulted in the synthesis of a cytoplasmic protein with a molecular weight of 35,000. The enzyme was purified, and its kinetic parameters were determined. Its Km values for glucose and ATP were 0.78 and 3.76 mM, respectively. Its Vmax was 158 U/mg of protein. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression. Under all conditions tested, only growth on glucose reduced the expression of glk by about 50%. A fruR mutation slightly increased the expression of glk-lacZ, whereas the overexpression of plasmid-encoded fruR+ weakly decreased expression. A FruR consensus binding motif was found 123 bp upstream of the potential transcriptional start site of glk. Overexpression of glk interfered with the expression of the maltose system. Repression was strongest in strains that exhibited constitutive mal gene expression due to endogenous induction and, in the absence of a functional MalK protein, the ATP-hydrolyzing subunit of the maltose transport system. It was least effective in wild-type strains growing on maltose or in strains constitutive for the maltose system due to a mutation in malT rendering the mal gene expression independent of inducer. This demonstrates that free internal glucose plays an essential role in the formation of the endogenous inducer of the maltose system.

Characterization of a cytoplasmic trehalase of Escherichia coli.

Escherichia coli can synthesize trehalose in response to osmotic stress and is able to utilize trehalose as a carbon source. The pathway of trehalose utilization is different at low and high osmolarity. At high osmolarity, a periplasmic trehalase (TreA) is induced that hydrolyzes trehalose in the periplasm to glucose. Glucose is then taken up by the phosphotransferase system. At low osmolarity, trehalose is taken up by a trehalose-specific enzyme II of the phosphotransferase system as trehalose-6-phosphate and then is hydrolyzed to glucose and glucose-6-phosphate. Here we report a novel cytoplasmic trehalase that hydrolyzes trehalose to glucose. treF, the gene encoding this enzyme, was cloned under ara promoter control. The enzyme (TreF) was purified from extracts of an overexpressing strain and characterized biochemically. It is specific for trehalose exhibiting a Km of 1.9 mM and a Vmax of 54 micromol of trehalose hydrolyzed per min per mg of protein. The enzyme is monomeric, exhibits a broad pH optimum at 6.0, and shows no metal dependency. TreF has a molecular weight of 63,703 (549 amino acids) and is highly homologous to TreA. The nonidentical amino acids of TreF are more polar and more acidic than those of TreA. The expression of treF as studied by the expression of a chromosomal treF-lacZ fusion is weakly induced by high osmolarity of the medium and is partially dependent on RpoS, the stationary-phase sigma factor. Mutants producing 17-fold more TreF than does the wild type were isolated.

Improved method for the preparative synthesis of labeled trehalose of high specific activity by Escherichia coli.

We report an improvement of a published procedure using Escherichia coli to synthesize 14C-labeled trehalose from [14C]glucose (B. Brand and W. Boos, Appl. Environ. Microbiol. 55:2414-2415, 1989). Instead of inducing the expression of the trehalose-synthesizing enzymes encoded by the chromosomal genes otsAB by high osmolarity, we now induce their expression from a plasmid under normal growth conditions by the addition of IPTG (isopropyl-beta-D-thiogalactopyranoside). Instead of using a pgi zwf double mutant to prevent glucose utilization, we use a pgi::Tn10 insertion only. In addition to being defective in treA, which encodes a periplasmic trehalase, the strain is now also defective in treF, which encodes a newly discovered cytoplasmic trehalase. This strain is genetically stable; it has no growth defects; and after induction with IPTG, it will transform [14C]glucose to [14C]trehalose in minimal medium without any carbon source under aerobic conditions at a rate of 3 nmol/min/10(9) cells. With the improved method, the overall yield of trehalose from glucose is about 80% and the process takes place without dilution of the specific radioactivity of the glucose residues. The accumulated trehalose is extracted from the bacteria by 70% hot ethanol and can easily be purified radiochemically by chromatographic techniques.

Molecular analysis of treB encoding the Escherichia coli enzyme II specific for trehalose.

A gene bank of partially Sau3A-digested Escherichia coli DNA ligated in plasmid pBR322 was screened for the ability to complement a mutant unable to metabolize trehalose at low osmolarity. The resulting plasmid was shown to contain the genes encoding transport (treB) and metabolic (treC) functions. The complementing DNA region was sequenced and shown to contain an operon of two genes, with treB as the promoter proximal gene and with treC as the promoter distal gene. The transcriptional start point was determined, and one major transcript was detected. The control region of the operon was found to contain consensus binding motifs for the cyclic AMP-catabolite activator protein complex and for a specific repressor protein whose gene, treR, is located immediately upstream of treB, being transcribed in the same direction as treB treC. The products of both genes could be expressed in minicells in which TreB revealed itself as a protein with an apparent molecular weight of 42,000. The gene product of treB consists of 485 amino acids with a calculated molecular weight of 52,308. It showed high homology to enzymes IIScr of enteric bacteria specific for the uptake of sucrose and encoded by plasmid pUR400 of enteric bacteria. Like enzyme IIScr, enzyme IITre belongs to the EIIBC domain type and lacks a covalently bound EIIA domain. Instead, enzyme IITre-mediated phosphorylation of trehalose requires the activity of enzyme IIAGlc, a component of the major glucose transport system.