Biomedical subjects

W Boos

Publications and source records attributed to W Boos.

At least 19 recordsLinked to original sources

PubMed2002

Crystallization and preliminary X-ray analysis of the trehalose/maltose ABC transporter MalFGK2 from Thermococcus litoralis.

Trehalose and maltose uptake in the hyperthermophilic archaeon Thermococcus litoralis is mediated by an ABC transport system. The heterotetrameric transport complex MalFGK(2), consisting of two membrane-spanning subunits and two copies of an ATP-binding cassette protein, has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 106.5, b = 150.5, c = 170.1 A, beta = 107.8 degrees. A native data set has been obtained at a resolution of 5 A.

Archaeal Proteins↗

PubMed2001

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.

ATP-Binding Cassette Transporters↗

PubMed2001

Purification and characterization of the heterologously expressed trehalose/maltose ABC transporter complex of the hyperthermophilic archaeon Thermococcus litoralis.

We report the purification of the maltose/trehalose transporter complex MalFGK of the hyperthermophilic archaeon Thermococcus litoralis. The complex was expressed in Escherichia coli, solubilized in dodecyl maltoside and purified with the aid of a histidine tag on one of the membrane proteins. One hundred grams of cells yielded 3 mg of pure complex. The final product showed ATPase activity at 70 degrees C and was soluble at low detergent concentration. ATPase activity was not due to dissociation of the MalK subunit from the integral membrane proteins MalF and MalG but could not be further stimulated by trehalose/maltose binding protein (TMBP), be it the native protein as isolated from T. litoralis or the soluble engineered protein. The purified native TMBP was identified as a glycoprotein.

ATP-Binding Cassette Transporters↗

PubMed2000

Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis.

The members of the ABC transporter family transport a wide variety of molecules into or out of cells and cellular compartments. Apart from a translocation pore, each member possesses two similar nucleoside triphosphate-binding subunits or domains in order to couple the energy-providing reaction with transport. In the maltose transporter of several Gram-negative bacteria and the archaeon Thermo coccus litoralis, the nucleoside triphosphate-binding subunit contains a C-terminal regulatory domain. A dimer of the subunit is attached cytoplasmically to the translocation pore. Here we report the crystal structure of this dimer showing two bound pyrophosphate molecules at 1.9 A resolution. The dimer forms by association of the ATPase domains, with the two regulatory domains attached at opposite poles. Significant deviation from 2-fold symmetry is seen at the interface of the dimer and in the regions corresponding to those residues known to be in contact with the translocation pore. The structure and its relationship to function are discussed in the light of known mutations from the homologous Escherichia coli and Salmonella typhimurium proteins.

ATP-Binding Cassette Transporters↗

PubMed2000

Mathematical treatment of transport data of bacterial transport systems to estimate limitation in diffusion through the outer membrane.

Bacterial transport systems are traditionally treated as enzymes exhibiting a saturable binding site giving rise to an apparent K(m)of transport, whereas the maximal rate of transport is regarded equivalent to the V(max)of enzymatic reactions. Thus, the Michaelis-Menten theory is usually applied in the analysis of transport data and K(m)and V(max)are derived from the treatment of data obtained from the rate of transport at varying substrate concentrations. Such an analysis tacitly assumes that the substrate recognition site of the transport system is freely accessible to substrate. However, this is not always the case. In systems endowed with high affinity in the micro M range or those recognizing large substrates or those exhibiting high V(max), the diffusion through the outer membrane may become rate determining, particularly at low external substrate concentrations. In such a situation the dependence of the overall rate of transport (from the medium into the cytoplasm) on the substrate concentration in the medium will no longer follow Michaelis-Menten kinetics. By analysing the deviation of transport data from the corresponding ideal Michaelis-Menten plot we developed a method that allows us to determine diffusion limitation through the outer membrane. The method allows us to find the correct K(m)of the transport system functioning at the inner membrane even under conditions of strong diffusion limitation through the outer membrane. The model was tested and validified with the Escherichia coli binding protein-dependent ABC transporter for maltose. The corresponding systems for sn -glycerol-3-phospate of Escherichia coli and the alpha -cyclodextrin transport of Klebsiella oxitoca were used as test systems.

ATP-Binding Cassette Transporters↗

PubMed2000

Signal transduction between a membrane-bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli.

The global regulator Mlc controls several genes implicated in sugar utilization systems, notably the phosphotransferase system (PTS) genes, ptsG, manXYZ and ptsHI, as well as the malT activator. No specific low molecular weight inducer has been identified that can inactivate Mlc, but its activity appeared to be modulated by transport of glucose via Enzyme IICB(Glc) (PtsG). Here we demonstrate that inactivation of Mlc is achieved by sequestration of Mlc to membranes containing dephosphorylated Enzyme IICB(Glc). We show that Mlc binds specifically to membrane fractions which carry PtsG and that excess Mlc can inhibit Enzyme IICB(Glc) phosphorylation by the general PTS proteins and also Enzyme IICB(Glc)-mediated phosphorylation of alpha-methylglucoside. Binding of Mlc to Enzyme IICB(Glc) in vitro required the IIB domain and the IIC-B junction region. Moreover, we show that these same regions are sufficient for Mlc regulation in vivo, via cross-dephosphorylation of IIB(Glc) during transport of other PTS sugars. The control of Mlc activity by sequestration to a transport protein represents a novel form of signal transduction in gene regulation.

Bacterial Proteins↗

PubMed2000

X-ray structure of MalY from Escherichia coli: a pyridoxal 5'-phosphate-dependent enzyme acting as a modulator in mal gene expression.

MalY represents a bifunctional pyridoxal 5'-phosphate-dependent enzyme acting as a beta-cystathionase and as a repressor of the maltose regulon. Here we present the crystal structures of wild-type and A221V mutant protein. Each subunit of the MalY dimer is composed of a large pyridoxal 5'-phosphate-binding domain and a small domain similar to aminotransferases. The structural alignment with related enzymes identifies residues that are generally responsible for beta-lyase activity and depicts a unique binding mode of the pyridoxal 5'-phosphate correlated with a larger, more flexible substrate-binding pocket. In a screen for MalY mutants with reduced mal repressor properties, mutations occurred in three clusters: I, 83-84; II, 181-189 and III, 215-221, which constitute a clearly distinguished region in the MalY crystal structure far away from the cofactor. The tertiary structure of one of these mutants (A221V) demonstrates that positional rearrangements are indeed restricted to regions I, II and III. Therefore, we propose that a direct protein-protein interaction with MalT, the central transcriptional activator of the maltose system, underlies MalY-dependent repression of the maltose system.

Bacterial Proteins↗

PubMed2000

Learning new tricks from an old dog: MalT of the Escherichia coli maltose system is part of a complex regulatory network.

The regulation of the maltose system in Escherichia coli has traditionally been viewed as a simple positive feedback loop. Today, we know that there are cross connections to several, seemingly unrelated, metabolic pathways. MalT, the central activator of the mal genes, is the key element in this complex regulatory network and integrates the different signals to give an appropriate transcriptional response.

Bacterial Proteins↗

PubMed2000

A new mechanism for the control of a prokaryotic transcriptional regulator: antagonistic binding of positive and negative effectors.

MalT, the transcriptional activator of the Escherichia coli maltose regulon, self-associates, binds promoter DNA and activates initiation of transcription only in the presence of ATP and maltotriose, the inducer. In vivo studies have revealed that MalT action is negatively controlled by the MalY protein. Using a biochemical approach, we analyse here the mechanism whereby MalY represses MalT activity. We show that MalY inhibits transcription activation by MalT in a purified transcription system. In vitro, a constitutive MalT variant (which is partially active in the absence of maltotriose) is less sensitive than wild-type MalT to repression by MalY, as observed in vivo. We demonstrate that MalY forms a complex with MalT only in the absence of maltotriose and that, conversely, MalY inhibits maltotriose binding by MalT. Together, these results establish that MalY acts directly upon MalT without the help of any factor, and that MalY is a negative effector of MalT competing with the inducer for MalT binding.

Bacterial Proteins↗

PubMed2000

Completion of the hook-basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription.

The flhDC operon of Salmonella typhimurium is the master control operon required for the expression of the entire flagellar regulon. The flagellar master operon was placed under the tetracycline-inducible promoter PtetA using the T-POP transposon. Cells containing this construct are motile in the presence of tetracycline and non-motile without inducer present. No flagella were visible under the electron microscope when cells were grown without inducer. The class 1, class 2 and class 3 promoters of the flagellar regulon are temporally regulated. After addition of tetracycline, the class 1 flhDC operon was transcribed immediately. Transcription of flgM (which is transcribed from both class 2 and class 3 promoters) began 15 min after induction. At 20 min after induction, the class 2 fliA promoter became active and intracellular FliA protein levels increased; at 30 min after induction, the class 3 fliC promoter was activated. Induction of fliC gene expression coincides with the appearance of FlgM anti-sigma factor in the growth medium. This also coincides with the completion of hook-basal body structures. Rolling cells first appeared 35 min after induction, and excess hook protein (FlgE) was also found in the growth medium at this time. At 45 min after induction, nascent flagellar filaments became visible in electron micrographs and over 40% of the cells exhibited some swimming behaviour. Multiple flagella assemble and grow on individual cells after induction of the master operon. These results confirm that the flagellar regulatory hierarchy of S. typhimurium is temporally regulated after induction. Both FlgM secretion and class 3 gene expression occur upon completion of the hook-basal body structure.

Antiporters↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed1999

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.

ATP-Binding Cassette Transporters↗

PubMed1999

Automated sampling device for monitoring intracellular metabolite dynamics.

An automated sampling device coupled to a stirred tank reactor was developed for monitoring intracellular metabolite dynamics. Sample flasks fixed in transport magazines were moved by a step engine in a way that each sample flask was filled within 220 ms, resulting in a sampling rate of 4.5 s-1. Rapid inactivation of the metabolism was achieved by spraying the samples into 60% methanol at -50 degrees C. After centrifugation of the quenched cells at -20 degrees C the metabolites were extracted with perchloric acid and analyzed biochemically or with HPLC. The automated sampling device was applied for investigation of the intracellular metabolite dynamics of glycolysis in Escherichia coli after rapid glucose addition to a glucose-limited steady-state culture. For the first time oscillations of intracellular metabolite concentrations like glucose-6-phosphate, phosphoenolpyruvate, glyceraldehyde 3-phosphate, dihydroxyacetonphosphate, 3-phosphoglycerate, and pyruvate were quantified on a subseconds to seconds scale in E. coli. As an example, the kinetics of the decomposition of fructose 1, 6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetonphosphate were investigated by use of a well-known mechanistic kinetic model and the measured in vivo metabolite dynamics.

Biosensing Techniques↗

PubMed1999

The role of the trehalose system in regulating the maltose regulon of Escherichia coli.

The maltose regulon consists of 10 genes encoding an ABC transporter for maltose and maltodextrins as well as enzymes necessary for their degradation. MalK, the energy-transducing subunit of the transport system, acts phenotypically as a repressor of MalT, the transcriptional activator of the mal genes. Using MacConkey maltose indicator plates we isolated an insertion mutation that strongly reduced the repressing effect of overproduced MalK. The insertion had occurred in treR encoding the repressor of the trehalose system. The loss of TreR function led to derepression of treB encoding an enzymeIITre of the PTS for trehalose and of treC encoding TreC, the cytoplasmic trehalose-6-phosphate hydrolase. Further analysis revealed that maltose can enter the cell by facilitated diffusion through enzymeIITre, thus causing induction of the maltose system. In addition, derepression of TreC by itself caused induction of the maltose system, and a mutant lacking TreC was reduced in the uninduced level of mal gene expression indicating synthesis of endogenous inducer by TreC. Extracts containing TreC transformed [14C]-maltose into another 14C-labelled compound (preliminarily identified as maltose 1-phosphate) that is likely to be an alternative inducer of the maltose system.

ATP-Binding Cassette Transporters↗

PubMed1999

Glycerol-3-phosphate-mediated repression of malT in Escherichia coli does not require metabolism, depends on enzyme IIAGlc and is mediated by cAMP levels.

malT encodes the central activator of the maltose system in Escherichia coli, a gene that is typically under positive control of the cAMP/CAP catabolite repression system. When cells were grown in tryptone broth, the addition of glycerol reduced malT expression two- to threefold. Phosphorylation of glycerol to glycerol-3-phosphate (G3P) was necessary for this repression, but further metabolism to dihydroxyacetone phosphate was not. Mutants lacking adenylate cyclase and harbouring a crp* mutation (synthesizing a cAMP receptor protein that is independent of cAMP) no longer repressed a transcriptional malT-lacZ fusion but still repressed a translational malT-lacZ fusion. Similar results were obtained with a mutant lacking enzyme IIAGlc. For the translational fusion (in a cya crp* genetic background) to be repressed by glycerol, a drop to pH 5 of the growth medium was necessary. Thus, while transcriptional repression by glycerol requires enzyme IIAGlc, cAMP and CAP, pH-mediated translational repression is cAMP independent. Other sugars that are not transported by the phosphotransferase system, most notably D-xylose, showed the same effect as glycerol.

Artificial Gene Fusion↗

PubMed1999

Maltose metabolism in the hyperthermophilic archaeon Thermococcus litoralis: purification and characterization of key enzymes.

Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-alpha-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-alpha-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an alpha-glucosidase, a p-nitrophenyl-alpha-D-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98 degrees C), of 66 U/mg. A Km of 0.46 mM was determined with heptaose as the substrate at 60 degrees C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5'-phosphate is conserved in the T. litoralis enzyme.

Amino Acid Sequence↗

PubMed1998

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.

Bacterial Proteins↗

PubMed1998

Enzymatic preparation of radiolabeled linear maltodextrins and cyclodextrins of high specific activity from [14C] maltose using amylomaltase, cyclodextrin glucosyltransferase and cyclodextrinase.

Radiolabeled linear and cyclic maltodextrins of high specific radioactivity were prepared using enzymes involved in maltodextrin metabolism. 14C-Labeled maltose was the starting material yielding products of identical specific radioactivity with respect to glucosyl residues. The enzymatic steps involved: i) Formation of linear 14C-labeled maltodexrins (< maltooctaose) using amylomaltase from Escherischia coli; ii) Cyclisation to alpha-cyclodextrin using cyclodextrin-glucosyltransferase of Kiebsiella oxytoca M5a1; iii) Removal of the remaining linear dextrins by amyloglucosidase. The products were purified by paper chromatography, or maltohexaose was specifically obtained from purified alpha-cyclodextrin by the action of cyclodextrinase of K. oxytoca M5a1.

Autoradiography↗