@article {1577, title = {Correlated mutation analyses on super-family alignments reveal functionally important residues.}, journal = {Proteins}, volume = {76}, year = {2009}, month = {2009 Aug 15}, pages = {608-16}, abstract = {

Correlated mutation analyses (CMA) on multiple sequence alignments are widely used for the prediction of the function of amino acids. The accuracy of CMA-based predictions is mainly determined by the number of sequences, by their evolutionary distances, and by the quality of the alignments. These criteria are best met in structure-based sequence alignments of large super-families. So far, CMA-techniques have mainly been employed to study the receptor interactions. The present work shows how a novel CMA tool, called Comulator, can be used to determine networks of functionally related residues in enzymes. These analyses provide leads for protein engineering studies that are directed towards modification of enzyme specificity or activity. As proof of concept, Comulator has been applied to four enzyme super-families: the isocitrate lyase/phoshoenol-pyruvate mutase super-family, the hexokinase super-family, the RmlC-like cupin super-family, and the FAD-linked oxidases super-family. In each of those cases networks of functionally related residue positions were discovered that upon mutation influenced enzyme specificity and/or activity as predicted. We conclude that CMA is a powerful tool for redesigning enzyme activity and selectivity.

}, keywords = {Algorithms, Computational Biology, Glucose-6-Phosphate Isomerase, Hexokinase, Isocitrate Lyase, Models, Molecular, Mutagenesis, Oxidoreductases, Oxidoreductases Acting on CH-CH Group Donors, Protein Structure, Secondary, Software}, issn = {1097-0134}, doi = {10.1002/prot.22374}, author = {Kuipers, Remko K P and Joosten, Henk-Jan and Verwiel, Eugene and Paans, Sjoerd and Akerboom, Jasper and van der Oost, John and Leferink, Nicole G H and van Berkel, Willem J H and Vriend, Gert and Schaap, Peter J} } @article {1581, title = {Structural insight into substrate binding and catalysis of a novel 2-keto-3-deoxy-D-arabinonate dehydratase illustrates common mechanistic features of the FAH superfamily.}, journal = {Journal of Molecular Biology}, volume = {379}, year = {2008}, month = {2008 May 30}, pages = {357-71}, abstract = {

The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-D-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzyme has been solved to 2.1 A resolution. The enzyme forms an oval-shaped ring of four subunits, each consisting of an N-terminal domain with a four-stranded beta-sheet flanked by two alpha-helices, and a C-terminal catalytic domain with a fumarylacetoacetate hydrolase (FAH) fold. Crystal structures of complexes of the enzyme with magnesium or calcium ions and either a substrate analog 2-oxobutyrate, or the aldehyde enzyme product 2,5-dioxopentanoate revealed that the divalent metal ion in the active site is coordinated octahedrally by three conserved carboxylate residues, a water molecule, and both the carboxylate and the oxo groups of the substrate molecule. An enzymatic mechanism for base-catalyzed dehydration is proposed on the basis of the binding mode of the substrate to the metal ion, which suggests that the enzyme enhances the acidity of the protons alpha to the carbonyl group, facilitating their abstraction by glutamate 114. A comprehensive structural comparison of members of the FAH superfamily is presented and their evolution is discussed, providing a basis for functional investigations of this largely unexplored protein superfamily.

}, keywords = {Amino Acid Sequence, Animals, Archaeal Proteins, Binding Sites, Catalysis, Crystallography, Humans, Hydro-Lyases, Hydrolases, Magnesium, Models, Molecular, Molecular Sequence Data, Molecular Structure, Protein Binding, Protein Structure, Protein Subunits, Quaternary, Secondary, Sequence Alignment, Substrate Specificity, Sulfolobus solfataricus, Tertiary, X-Ray}, issn = {1089-8638}, doi = {10.1016/j.jmb.2008.03.064}, author = {Brouns, Stan J J and Barends, Thomas R M and Worm, Petra and Akerboom, Jasper and Turnbull, Andrew P and Salmon, Laurent and van der Oost, John} } @article {1585, title = {Biochemical and structural exploration of the catalytic capacity of Sulfolobus KDG aldolases.}, journal = {The Biochemical Journal}, volume = {403}, year = {2007}, month = {2007 May 1}, pages = {421-30}, abstract = {

Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.

}, keywords = {Aldehyde-Lyases, Aldehydes, Binding Sites, Cloning, Crystallization, Crystallography, Escherichia coli, Models, Molecular, Pyruvates, Substrate Specificity, Sulfolobus, X-Ray}, issn = {1470-8728}, doi = {10.1042/BJ20061419}, author = {Wolterink-van Loo, Suzanne and van Eerde, Andr{\'e} and Siemerink, Marco A J and Akerboom, Jasper and Dijkstra, Bauke W and van der Oost, John} } @article {1583, title = {Crystal structure and biochemical properties of the D-arabinose dehydrogenase from Sulfolobus solfataricus.}, journal = {Journal of Molecular Biology}, volume = {371}, year = {2007}, month = {2007 Aug 31}, pages = {1249-60}, abstract = {

Sulfolobus solfataricus metabolizes the five-carbon sugar d-arabinose to 2-oxoglutarate by an inducible pathway consisting of dehydrogenases and dehydratases. Here we report the crystal structure and biochemical properties of the first enzyme of this pathway: the d-arabinose dehydrogenase. The AraDH structure was solved to a resolution of 1.80 A by single-wavelength anomalous diffraction and phased using the two endogenous zinc ions per subunit. The structure revealed a catalytic and cofactor binding domain, typically present in mesophilic and thermophilic alcohol dehydrogenases. Cofactor modeling showed the presence of a phosphate binding pocket sequence motif (SRS-X2-H), which is likely to be responsible for the enzyme{\textquoteright}s preference for NADP+. The homo-tetrameric enzyme is specific for d-arabinose, l-fucose, l-galactose and d-ribose, which could be explained by the hydrogen bonding patterns of the C3 and C4 hydroxyl groups observed in substrate docking simulations. The enzyme optimally converts sugars at pH 8.2 and 91 degrees C, and displays a half-life of 42 and 26 min at 85 and 90 degrees C, respectively, indicating that the enzyme is thermostable at physiological operating temperatures of 80 degrees C. The structure represents the first crystal structure of an NADP+-dependent member of the medium-chain dehydrogenase/reductase (MDR) superfamily from Archaea.

}, keywords = {Amino Acid, Amino Acid Sequence, Binding Sites, Carbohydrates, Catalytic Domain, Crystallization, Crystallography, Hydrogen-Ion Concentration, Molecular Conformation, Molecular Sequence Data, Protein Binding, Protein Structure, Quaternary, Sequence Homology, Sugar Alcohol Dehydrogenases, Sulfolobus solfataricus, Temperature, X-Ray}, issn = {0022-2836}, doi = {10.1016/j.jmb.2007.05.097}, author = {Brouns, Stan J J and Turnbull, Andrew P and Willemen, Hanneke L D M and Akerboom, Jasper and van der Oost, John} } @article {1582, title = {A global transcriptional regulator in Thermococcus kodakaraensis controls the expression levels of both glycolytic and gluconeogenic enzyme-encoding genes.}, journal = {The Journal of Biological Chemistry}, volume = {282}, year = {2007}, month = {2007 Nov 16}, pages = {33659-70}, abstract = {

We identified a novel regulator, Thermococcales glycolytic regulator (Tgr), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Tgr (TK1769) displays similarity (28\% identical) to Pyrococcus furiosus TrmB (PF1743), a transcriptional repressor regulating the trehalose/maltose ATP-binding cassette transporter genes, but is more closely related (67\%) to a TrmB paralog in P. furiosus (PF0124). Growth of a tgr disruption strain (Deltatgr) displayed a significant decrease in growth rate under gluconeogenic conditions compared with the wild-type strain, whereas comparable growth rates were observed under glycolytic conditions. A whole genome microarray analysis revealed that transcript levels of almost all genes related to glycolysis and maltodextrin metabolism were at relatively high levels in the Deltatgr mutant even under gluconeogenic conditions. The Deltatgr mutant also displayed defects in the transcriptional activation of gluconeogenic genes under these conditions, indicating that Tgr functions as both an activator and a repressor. Genes regulated by Tgr contain a previously identified sequence motif, the Thermococcales glycolytic motif (TGM). The TGM was positioned upstream of the Transcription factor B-responsive element (BRE)/TATA sequence in gluconeogenic promoters and downstream of it in glycolytic promoters. Electrophoretic mobility shift assay indicated that recombinant Tgr protein specifically binds to promoter regions containing a TGM. Tgr was released from the DNA when maltotriose was added, suggesting that this sugar is most likely the physiological effector. Our results strongly suggest that Tgr is a global transcriptional regulator that simultaneously controls, in response to sugar availability, both glycolytic and gluconeogenic metabolism in T. kodakaraensis via its direct binding to the TGM.

}, keywords = {Amino Acid, Amino Acid Motifs, Amino Acid Sequence, Escherichia coli, Genetic, Genome, Genotype, Gluconeogenesis, Glycolysis, Molecular Sequence Data, Multienzyme Complexes, NADH, NADPH Oxidoreductases, Oligonucleotide Array Sequence Analysis, Promoter Regions, Pyrococcus furiosus, Recombinant Proteins, Sequence Homology, Thermococcus, Transcription}, issn = {0021-9258}, doi = {10.1074/jbc.M703424200}, author = {Kanai, Tamotsu and Akerboom, Jasper and Takedomi, Shogo and van de Werken, Harmen J G and Blombach, Fabian and van der Oost, John and Murakami, Taira and Atomi, Haruyuki and Imanaka, Tadayuki} } @article {1584, title = {Purification, crystallization and preliminary crystallographic analysis of a GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus.}, journal = {Acta Crystallographica. Section F, Structural Biology and Crystallization Communications}, volume = {63}, year = {2007}, month = {2007 Mar 1}, pages = {239-41}, abstract = {

A predicted GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus, termed SsGBP, has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion technique in the presence of 0.05 M cadmium sulfate and 0.8 M sodium acetate pH 7.5. A single-wavelength anomalous dispersion data set was collected to a maximum resolution of 2.0 A using a single cadmium-incorporated crystal. The crystal form belongs to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 65.0

}, keywords = {Archaeal Proteins, Crystallization, Crystallography, GTP-Binding Proteins, Hot Temperature, Sulfolobus solfataricus, X-Ray}, issn = {1744-3091}, doi = {10.1107/S1744309107008500}, author = {Wu, Hao and Sun, Lei and Brouns, Stan J J and Fu, Sheng and Akerboom, Jasper and Li, Xuemei and van der Oost, John} } @article {1587, title = {Evidence supporting a cis-enediol-based mechanism for Pyrococcus furiosus phosphoglucose isomerase.}, journal = {Journal of Molecular Biology}, volume = {358}, year = {2006}, month = {2006 May 19}, pages = {1353-66}, abstract = {

The enzymatic aldose ketose isomerisation of glucose and fructose sugars involves the transfer of a hydrogen between their C1 and C2 carbon atoms and, in principle, can proceed through either a direct hydride shift or via a cis-enediol intermediate. Pyrococcus furiosus phosphoglucose isomerase (PfPGI), an archaeal metalloenzyme, which catalyses the interconversion of glucose 6-phosphate and fructose 6-phosphate, has been suggested to operate via a hydride shift mechanism. In contrast, the structurally distinct PGIs of eukaryotic or bacterial origin are thought to catalyse isomerisation via a cis-enediol intermediate. We have shown by NMR that hydrogen exchange between substrate and solvent occurs during the reaction catalysed by PfPGI eliminating the possibility of a hydride-shift-based mechanism. In addition, kinetic measurements on this enzyme have shown that 5-phospho-d-arabinonohydroxamate, a stable analogue of the putative cis-enediol intermediate, is the most potent inhibitor of the enzyme yet discovered. Furthermore, determination and analysis of crystal structures of PfPGI with bound zinc and the substrate F6P, and with a number of competitive inhibitors, and EPR analysis of the coordination of the metal ion within PfPGI, have suggested that a cis-enediol intermediate-based mechanism is used by PfPGI with Glu97 acting as the catalytic base responsible for isomerisation.

}, keywords = {Biomolecular, Catalytic Domain, Chemical, Crystallography, Electron Spin Resonance Spectroscopy, Enzyme Inhibitors, Glucose-6-Phosphate Isomerase, Hydrogen, Hydroxamic Acids, Isomerism, Kinetics, Macromolecular Substances, Metals, Models, Molecular, Nuclear Magnetic Resonance, Pyrococcus furiosus, Stereoisomerism, Substrate Specificity, Sugar Phosphates, X-Ray}, issn = {0022-2836}, doi = {10.1016/j.jmb.2006.03.015}, author = {Berrisford, John M and Hounslow, Andrea M and Akerboom, Jasper and Hagen, Wilfred R and Brouns, Stan J J and van der Oost, John and Murray, Iain A and Michael Blackburn, G and Waltho, Jonathan P and Rice, David W and Baker, Patrick J} } @article {1586, title = {Identification of a glycolytic regulon in the archaea Pyrococcus and Thermococcus.}, journal = {FEMS Microbiology Letters}, volume = {260}, year = {2006}, month = {2006 Jul}, pages = {69-76}, abstract = {

The glycolytic pathway of the hyperthermophilic archaea that belong to the order Thermococcales (Pyrococcus, Thermococcus and Palaeococcus) differs significantly from the canonical Embden-Meyerhof pathway in bacteria and eukarya. This archaeal glycolysis variant consists of several novel enzymes, some of which catalyze unique conversions. Moreover, the enzymes appear not to be regulated allosterically, but rather at transcriptional level. To elucidate details of the gene expression control, the transcription initiation sites of the glycolytic genes in Pyrococcus furiosus have been mapped by primer extension analysis and the obtained promoter sequences have been compared with upstream regions of non-glycolytic genes. Apart from consensus sequences for the general transcription factors (TATA-box and BRE) this analysis revealed the presence of a potential transcription factor binding site (TATCAC-N(5)-GTGATA) in glycolytic and starch utilizing promoters of P. furiosus and several thermococcal species. The absence of this inverted repeat in Pyrococcus abyssi and Pyrococcus horikoshii probably reflects that their reduced catabolic capacity does not require this regulatory system. Moreover, this phyletic pattern revealed a TrmB-like regulator (PF0124 and TK1769) which may be involved in recognizing the repeat. This Thermococcales glycolytic regulon, with more than 20 genes, is the largest regulon that has yet been described for Archaea.

}, keywords = {Base Sequence, Binding Sites, Genetic, Glycolysis, Molecular Sequence Data, Promoter Regions, Pyrococcus, Regulon, Thermococcus, Transcription Initiation Site}, issn = {0378-1097}, doi = {10.1111/j.1574-6968.2006.00292.x}, author = {van de Werken, Harmen J G and Verhees, Corn{\'e} H and Akerboom, Jasper and de Vos, Willem M and van der Oost, John} } @article {1588, title = {Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family.}, journal = {Nucleic Acids Research}, volume = {34}, year = {2006}, month = {2006 Mar 9}, pages = {1439-49}, abstract = {

The Lrp/AsnC family of transcriptional regulatory proteins is found in both archaea and bacteria. Members of the family influence cellular metabolism in both a global (Lrp) and specific (AsnC) manner, often in response to exogenous amino acid effectors. In the present study we have determined both the first bacterial and the highest resolution structures for members of the family. Escherichia coli AsnC is a specific gene regulator whose activity is triggered by asparagine binding. Bacillus subtilis LrpC is a global regulator involved in chromosome condensation. Our AsnC-asparagine structure is the first for a regulator-effector complex and is revealed as an octameric disc. Key ligand recognition residues are identified together with a route for ligand access. The LrpC structure reveals a stable octamer supportive of a topological role in dynamic DNA packaging. The structures yield significant clues to the functionality of Lrp/AsnC-type regulators with respect to ligand binding and oligomerization states as well as to their role in specific and global DNA regulation.

}, keywords = {Amino Acid Sequence, Asparagine, Bacillus subtilis, Bacterial, Bacterial Proteins, Escherichia coli Proteins, Gene Expression Regulation, Genetic, Ligands, Models, Molecular, Molecular Sequence Data, Sequence Alignment, Trans-Activators, Transcription, Transcription Factors}, issn = {1362-4962}, doi = {10.1093/nar/gkl009}, author = {Thaw, Paul and Sedelnikova, Svetlana E and Muranova, Tatyana and Wiese, Sebastian and Ayora, Sylvia and Alonso, Juan C and Brinkman, Arie B and Akerboom, Jasper and van der Oost, John and Rafferty, John B} } @article {1589, title = {Engineering a selectable marker for hyperthermophiles.}, journal = {The Journal of Biological Chemistry}, volume = {280}, year = {2005}, month = {2005 Mar 25}, pages = {11422-31}, abstract = {

Limited thermostability of antibiotic resistance markers has restricted genetic research in the field of extremely thermophilic Archaea and bacteria. In this study, we used directed evolution and selection in the thermophilic bacterium Thermus thermophilus HB27 to find thermostable variants of a bleomycin-binding protein from the mesophilic bacterium Streptoalloteichus hindustanus. In a single selection round, we identified eight clones bearing five types of double mutated genes that provided T. thermophilus transformants with bleomycin resistance at 77 degrees C, while the wild-type gene could only do so up to 65 degrees C. Only six different amino acid positions were altered, three of which were glycine residues. All variant proteins were produced in Escherichia coli and analyzed biochemically for thermal stability and functionality at high temperature. A synthetic mutant resistance gene with low GC content was designed that combined four substitutions. The encoded protein showed up to 17 degrees C increased thermostability and unfolded at 85 degrees C in the absence of bleomycin, whereas in its presence the protein unfolded at 100 degrees C. Despite these highly thermophilic properties, this mutant was still able to function normally at mesophilic temperatures in vivo. The mutant protein was co-crystallized with bleomycin, and the structure of the binary complex was determined to a resolution of 1.5 A. Detailed structural analysis revealed possible molecular mechanisms of thermostabilization and enhanced antibiotic binding, which included the introduction of an intersubunit hydrogen bond network, improved hydrophobic packing of surface indentations, reduction of loop flexibility, and alpha-helix stabilization. The potential applicability of the thermostable selection marker is discussed.

}, keywords = {Amino Acid Sequence, Bacterial, Bacterial Proteins, Biological Markers, Bleomycin, Carrier Proteins, Drug Resistance, Genetic Engineering, Hydrogen Bonding, Hydrophobic and Hydrophilic Interactions, Molecular Sequence Data, Protein Folding, Protein Structure, Secondary, Temperature, Thermus thermophilus}, issn = {0021-9258}, doi = {10.1074/jbc.M413623200}, author = {Brouns, Stan J J and Wu, Hao and Akerboom, Jasper and Turnbull, Andrew P and de Vos, Willem M and van der Oost, John} } @article {1590, title = {The structures of inhibitor complexes of Pyrococcus furiosus phosphoglucose isomerase provide insights into substrate binding and catalysis.}, journal = {Journal of Molecular Biology}, volume = {343}, year = {2004}, month = {2004 Oct 22}, pages = {649-57}, abstract = {

Pyrococcus furiosus phosphoglucose isomerase (PfPGI) is a metal-containing enzyme that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). The recent structure of PfPGI has confirmed the hypothesis that the enzyme belongs to the cupin superfamily and identified the position of the active site. This fold is distinct from the alphabetaalpha sandwich fold commonly seen in phosphoglucose isomerases (PGIs) that are found in bacteria, eukaryotes and some archaea. Whilst the mechanism of the latter family is thought to proceed through a cis-enediol intermediate, analysis of the structure of PfPGI in the presence of inhibitors has led to the suggestion that the mechanism of this enzyme involves the metal-dependent direct transfer of a hydride between C1 and C2 atoms of the substrate. To gain further insight in the reaction mechanism of PfPGI, the structures of the free enzyme and the complexes with the inhibitor, 5-phospho-d-arabinonate (5PAA) in the presence and absence of metal have been determined. Comparison of these structures with those of equivalent complexes of the eukaryotic PGIs reveals similarities at the active site in the disposition of possible catalytic residues. These include the presence of a glutamic acid residue, Glu97 in PfPGI, which occupies the same position relative to the inhibitor as that of the glutamate that is thought to function as the catalytic base in the eukaryal-type PGIs. These similarities suggest that aspects of the catalytic mechanisms of these two structurally unrelated PGIs may be similar and based on an enediol intermediate.

}, keywords = {Archaeal Proteins, Binding Sites, Catalysis, Crystallography, Dimerization, Fructosephosphates, Glucose-6-Phosphate, Glucose-6-Phosphate Isomerase, Manganese, Models, Molecular, Molecular Structure, Pentosephosphates, Protein Binding, Protein Conformation, Pyrococcus furiosus, X-Ray}, issn = {0022-2836}, doi = {10.1016/j.jmb.2004.08.061}, author = {Berrisford, John M and Akerboom, Jasper and Brouns, Stan and Sedelnikova, Svetlana E and Turnbull, Andrew P and van der Oost, John and Salmon, Laurent and Hardr{\'e}, Renaud and Murray, Iain A and Blackburn, G Michael and Rice, David W and Baker, Patrick J} } @article {1592, title = {Crystal structure of Pyrococcus furiosus phosphoglucose isomerase. Implications for substrate binding and catalysis.}, journal = {The Journal of Biological Chemistry}, volume = {278}, year = {2003}, month = {2003 Aug 29}, pages = {33290-7}, abstract = {

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization between d-fructose 6-phosphate and d-glucose 6-phosphate as part of the glycolytic pathway. PGI from the Archaea Pyrococcus furiosus (Pfu) was crystallized, and its structure was determined by x-ray diffraction to a 2-A resolution. Structural comparison of this archaeal PGI with the previously solved structures of bacterial and eukaryotic PGIs reveals a completely different structure. Each subunit of the homodimeric Pfu PGI consists of a cupin domain, for which the overall structure is similar to other cupin domain-containing proteins, and includes a conserved transition metal-binding site. Biochemical data on the recombinant enzyme suggests that Fe2+ is bound to Pfu PGI. However, as catalytic activity is not strongly influenced either by the replacement of Fe2+ by a range of transition metals or by the presence or absence of the bound metal ion, we suggest that the metal may not be directly involved in catalysis but rather may be implicated in substrate recognition.

}, keywords = {Amino Acid, Amino Acid Sequence, Binding Sites, Chemical, Crystallography, Dimerization, Fructosephosphates, Glucose-6-Phosphate, Glucose-6-Phosphate Isomerase, Ions, Iron, Metals, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Conformation, Protein Structure, Pyrococcus furiosus, Secondary, Selenomethionine, Sequence Homology, Substrate Specificity, Temperature, Tertiary, X-Ray, X-Ray Diffraction}, issn = {0021-9258}, doi = {10.1074/jbc.M305170200}, author = {Berrisford, John M and Akerboom, Jasper and Turnbull, Andrew P and de Geus, Daniel and Sedelnikova, Svetlana E and Staton, Ian and McLeod, Cameron W and Verhees, Corn{\'e} H and van der Oost, John and Rice, David W and Baker, Patrick J} } @article {1591, title = {Purification, crystallization and preliminary crystallographic analysis of phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus.}, journal = {Acta Crystallographica. Section D, Biological Crystallography}, volume = {59}, year = {2003}, month = {2003 Oct 19}, pages = {1822-3}, abstract = {

The glycolytic enzyme phosphoglucose isomerase catalyses the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus, which shows no sequence similarity to any known bacterial or eukaryotic phosphoglucose isomerase, has been cloned and overexpressed in Escherichia coli, purified and subsequently crystallized by the hanging-drop method of vapour diffusion using 1.6 M sodium citrate as the precipitant at pH 6.5. Multiple-wavelength anomalous dispersive X-ray data have been collected to a maximum resolution of 1.92 A on a single selenomethionine-incorporated crystal. This crystal belongs to space group C2, with approximate unit-cell parameters a = 84.7

}, keywords = {Crystallization, Crystallography, Escherichia coli, Glucose-6-Phosphate Isomerase, Protein Conformation, Pyrococcus furiosus, Recombinant Proteins, Selenomethionine, X-Ray}, issn = {0907-4449}, author = {Akerboom, Jasper and Turnbull, Andrew P and Hargreaves, David and Fisher, Martin and de Geus, Daniel and Sedelnikova, Svetlana E and Berrisford, John M and Baker, Patrick J and Verhees, Corn{\'e} H and van der Oost, John and Rice, David W} } @article {1593, title = {Molecular and biochemical characterization of a distinct type of fructose-1,6-bisphosphatase from Pyrococcus furiosus.}, journal = {Journal of Bacteriology}, volume = {184}, year = {2002}, month = {2002 Jun}, pages = {3401-5}, abstract = {

The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a K(m) of 0.32 mM and a V(max) of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li(+) (50\% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.

}, keywords = {Amino Acid Sequence, Archaeal Proteins, Cloning, DNA, Fructose-Bisphosphatase, Molecular, Molecular Sequence Data, Pyrococcus furiosus, Sequence Analysis, Substrate Specificity, Temperature}, issn = {0021-9193}, author = {Verhees, Corn{\'e} H and Akerboom, Jasper and Schiltz, Emile and de Vos, Willem M and van der Oost, John} }