Biomedical subjects

John van der Oost

Publications and source records attributed to John van der Oost.

At least 19 recordsLinked to original sources

PubMed2007

Biochemical and structural exploration of the catalytic capacity of Sulfolobus KDG aldolases.

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.

Aldehyde-Lyases↗

PubMed2006

Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry.

We modified and optimized a first generation quadrupole time-of-flight (Q-TOF) 1 to perform tandem mass spectrometry on macromolecular protein complexes. The modified instrument allows isolation and subsequent dissociation of high-mass protein complexes through collisions with argon molecules. The modifications of the Q-TOF 1 include the introduction of (1) a flow-restricting sleeve around the first hexapole ion bridge, (2) a low-frequency ion-selecting quadrupole, (3) a high-pressure hexapole collision cell, (4) high-transmission grids in the multicomponent ion lenses, and (5) a low repetition rate pusher. Using these modifications, we demonstrate the experimental isolation of ions up to 12 800 mass-to-charge units and detection of product ions up to 38 150 Da, enabling the investigation of the gas-phase stability, protein complex topology, and quaternary structure of protein complexes. Some of the data reveal a so-far unprecedented new mechanism in gas-phase dissociation of protein oligomers whereby a tetramer complex dissociates into two dimers. These data add to the current debate whether gas-phase structures of protein complexes do retain some of the structural features of the corresponding species in solution. The presented low-cost modifications on a Q-TOF 1 instrument are of interest to everyone working in the fields of macromolecular mass spectrometry and more generic structural biology.

Alcohol Oxidoreductases↗

PubMed2006

Fortuitous structure determination of 'as-isolated' Escherichia coli bacterioferritin in a novel crystal form.

Escherichia coli bacterioferritin was serendipitously crystallized in a novel cubic crystal form and its structure could be determined to 2.5 A resolution despite a high degree of merohedral twinning. This is the first report of crystallographic data on 'as-isolated' E. coli bacterioferritin. The ferroxidase active site contains positive difference density consistent with two metal ions that had co-purified with the protein. X-ray fluorescence studies suggest that the metal composition is different from that of previous structures and is a mix of zinc and native iron ions. The ferroxidase-centre configuration displays a similar flexibility as previously noted for other bacterioferritins.

Bacterial Proteins↗

PubMed2006

Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment.

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that D-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a D-arabinose dehydrogenase, a D-arabinonate dehydratase, a novel 2-keto-3-deoxy-D-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.

Arabinose↗

PubMed2006

CprK crystal structures reveal mechanism for transcriptional control of halorespiration.

Halorespiration is a bacterial respiratory process in which haloorganic compounds act as terminal electron acceptors. This process is controlled at transcriptional level by CprK, a member of the ubiquitous CRP-FNR family. Here we present the crystal structures of oxidized CprK in presence of the ligand ortho-chlorophenolacetic acid and of reduced CprK in absence of this ligand. These structures reveal that highly specific binding of chlorinated, rather than the corresponding non-chlorinated, phenolic compounds in the NH(2)-terminal beta-barrels causes reorientation of these domains with respect to the central alpha-helix at the dimer interface. Unexpectedly, the COOH-terminal DNA-binding domains dimerize in the non-DNA binding state. We postulate the ligand-induced conformational change allows formation of interdomain contacts that disrupt the DNA domain dimer interface and leads to repositioning of the helix-turn-helix motifs. These structures provide a structural framework for further studies on transcriptional control by CRP-FNR homologs in general and of halorespiration regulation by CprK in particular.

Bacterial Proteins↗

PubMed2006

Evidence supporting a cis-enediol-based mechanism for Pyrococcus furiosus phosphoglucose isomerase.

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.

Catalytic Domain↗

PubMed2006

Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family.

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.

Amino Acid Sequence↗

PubMed2006

Influence of temperature on the production of an archaeal thermoactive alcohol dehydrogenase from Pyrococcus furiosus with recombinant Escherichia coli.

The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD600 of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28 degrees C, heat shock of the culture from 37 to 42 degrees C and from 37 to 45 degrees C, and variation of time of induction (induction at an OD600 of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42 degrees C, and IPTG induction of the adhC expression at an OD600 of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular.

Alcohol Dehydrogenase↗

PubMed2006

Reconstruction of central carbon metabolism in Sulfolobus solfataricus using a two-dimensional gel electrophoresis map, stable isotope labelling and DNA microarray analysis.

In the last decade, an increasing number of sequenced archaeal genomes have become available, opening up the possibility for functional genomic analyses. Here, we reconstructed the central carbon metabolism in the hyperthermophilic crenarchaeon Sulfolobus solfataricus (glycolysis, gluconeogenesis and tricarboxylic acid cycle) on the basis of genomic, proteomic, transcriptomic and biochemical data. A 2-DE reference map of S. solfataricus grown on glucose, consisting of 325 unique ORFs in 255 protein spots, was created to facilitate this study. The map was then used for a differential expression study based on (15)N metabolic labelling (yeast extract + tryptone-grown cells (YT) vs. glucose-grown cells (G)). In addition, the expression ratio of the genes involved in carbon metabolism was studied using DNA microarrays. Surprisingly, only 3 and 14% of the genes and proteins, respectively, involved in central carbon metabolism showed a greater than two-fold change in expression level. All results are discussed in the light of the current understanding of central carbon metabolism in S. solfataricus and will help to obtain a system-wide understanding of this organism.

Archaeal Proteins↗

PubMed2006

Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2.

Using a comparative genomics approach, a copper resistance gene cluster has been identified in multiple archaeal genomes. The cop cluster is predicted to encode a metallochaperone (CopM), a P-type copper-exporting ATPase (CopA) and a novel, archaea-specific transcriptional regulator (CopT) which might control the expression of the cop genes. Sequence analysis revealed that CopT has an N-terminal DNA-binding helix-turn-helix domain and a C-terminal TRASH domain; TRASH is a novel domain which has recently been proposed to be uniquely involved in metal-binding in sensors, transporters and trafficking proteins in prokaryotes. The present study describes the molecular characterization of the cop gene cluster in the thermoacidophilic crenarchaeon Sulfolobus solfataricus. The polycistronic copMA transcript was found to accumulate in response to growth-inhibiting copper concentrations, whereas copT transcript abundance appeared to be constitutive. DNA-binding assays revealed that CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the copMA gene cluster.

Amino Acid Sequence↗

PubMed2006

Two novel conjugative plasmids from a single strain of Sulfolobus.

Two conjugative plasmids (CPs) were isolated and characterized from the same 'Sulfolobus islandicus' strain, SOG2/4. The plasmids were separated from each other and transferred into Sulfolobus solfataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number and is stably maintained (pSOG2). Plasmid pSOG2 is the first Sulfolobus CP found to have these characteristics. The genomes of both pSOG plasmids have been sequenced and were compared to each other and the available Sulfolobus CPs. Interestingly, apart from a very well-conserved core, 70 % of the pSOG1 and pSOG2 genomes is largely different and composed of a mixture of genes that often resemble counterparts in previously described Sulfolobus CPs. However, about 20 % of the predicted genes do not have known homologues, not even in other CPs. Unlike pSOG1, pSOG2 does not contain a gene for the highly conserved PlrA protein nor for obvious homologues of partitioning proteins. Unlike pNOB8 and pKEF9, both pSOG plasmids lack the so-called clustered regularly interspaced short palindrome repeats (CRISPRs). The sites of recombination between the two genomes can be explained by the presence of recombination motifs previously identified in other Sulfolobus CPs. Like other Sulfolobus CPs, the pSOG plasmids possess a gene encoding an integrase of the tyrosine recombinase family. This integrase probably mediates plasmid site-specific integration into the host chromosome at the highly conserved tRNA(Glu) loci.

Base Sequence↗

PubMed2006

Identification of a glycolytic regulon in the archaea Pyrococcus and Thermococcus.

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.

Base Sequence↗

PubMed2006

Production and characterization of a thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

The gene encoding a threonine dehydrogenase (TDH) has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The Pf-TDH protein has been functionally produced in Escherichia coli and purified to homogeneity. The enzyme has a tetrameric conformation with a molecular mass of approximately 155 kDa. The catalytic activity of the enzyme increases up to 100 degrees C, and a half-life of 11 min at this temperature indicates its thermostability. The enzyme is specific for NAD(H), and maximal specific activities were detected with L-threonine (10.3 U x mg(-1)) and acetoin (3.9 U x mg(-1)) in the oxidative and reductive reactions, respectively. Pf-TDH also utilizes L-serine and D-threonine as substrate, but could not oxidize other L-amino acids. The enzyme requires bivalent cations such as Zn2+ and Co2+ for activity and contains at least one zinc atom per subunit. Km values for L-threonine and NAD+ at 70 degrees C were 1.5 mm and 0.055 mm, respectively.

Alcohol Oxidoreductases↗

PubMed2006

Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily.

The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a molecular mass of 32 kDa. The catalytic activity of the enzyme increases up to 100 degrees C, and a half-life value of 130 min at this temperature indicates its high thermostability. AdhD exhibits a broad substrate specificity with, in general, a preference for the reduction of ketones (pH optimum, 6.1) and the oxidation of secondary alcohols (pH optimum, 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl-acetoin (22.5 U/mg) in the oxidative and reductive reactions, respectively. Gas chromatrography analysis indicated that AdhD produced mainly (S)-2-pentanol (enantiomeric excess, 89%) when 2-pentanone was used as substrate. The physiological role of AdhD is discussed.

Alcohol Dehydrogenase↗

PubMed2006

Identification of a novel alpha-galactosidase from the hyperthermophilic archaeon Sulfolobus solfataricus.

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable alpha-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for alpha-linked galactosides, which are optimally hydrolyzed at pH 5 and 90 degrees C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable alpha-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.

Enzyme Activation↗

PubMed2006

Characterization of CprK1, a CRP/FNR-type transcriptional regulator of halorespiration from Desulfitobacterium hafniense.

The recently identified CprK branch of the CRP (cyclic AMP receptor protein)-FNR (fumarate and nitrate reduction regulator) family of transcriptional regulators includes proteins that activate the transcription of genes encoding proteins involved in reductive dehalogenation of chlorinated aromatic compounds. Here we report the characterization of the CprK1 protein from Desulfitobacterium hafniense, an anaerobic low-G+C gram-positive bacterium that is capable of reductive dechlorination of 3-chloro-4-hydroxyphenylacetic acid (Cl-OHPA). The gene encoding CprK1 was cloned and functionally overexpressed in Escherichia coli, and the protein was subsequently purified to homogeneity. To investigate the interaction of CprK1 with three of its predicted binding sequences (dehaloboxes), we performed in vitro DNA-binding assays (electrophoretic mobility shift assays) as well as in vivo promoter probe assays. Our results show that CprK1 binds its target dehaloboxes with high affinity (dissociation constant, 90 nM) in the presence of Cl-OHPA and that transcriptional initiation by CprK1 is influenced by deviations in the dehaloboxes from the consensus TTAAT----ATTAA sequence. A mutant CprK1 protein was created by a Val-->Glu substitution at a conserved position in the recognition alpha-helix that gained FNR-type DNA-binding specificity, recognizing the TTGAT----ATCAA sequence (FNR box) instead of the dehaloboxes. CprK1 was subject to oxidative inactivation in vitro, most likely caused by the formation of an intermolecular disulfide bridge between Cys11 and Cys200. The possibility of redox regulation of CprK1 by a thiol-disulfide exchange reaction was investigated by using two Cys-->Ser mutants. Our results indicate that a Cys11-Cys200 disulfide bridge does not appear to play a physiological role in the regulation of CprK1.

Amino Acid Sequence↗

PubMed2005

Temperature-dependent structural and functional features of a hyperthermostable enzyme using elastic neutron scattering.

The dynamic behavior of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus was investigated using elastic neutron scattering. The temperature dependence of the atomic motions was correlated with conformational and functional characteristics of the enzyme. The onset of biological function at temperatures higher than approximately 25 degrees C (the hyperthermostable enzyme is essentially inactive at room temperature) was associated with a dynamical transition in the anharmonic motions domain. This transition from the nonactive to the enzymatically active conformation involved structurally similar conformational substates in the energy landscape. From the mean-square displacement of the protein atoms, the molecular flexibility and the effective force constants were calculated at different temperature zones. The results showed that the activity increases at higher temperatures where the intramolecular bonds are weakened and the overall rigidity of the protein is decreased. Further temperature increase resulted in significantly increased atomic fluctuations featuring heat denaturation of the protein.

Calorimetry↗

PubMed2005

The semi-phosphorylative Entner-Doudoroff pathway in hyperthermophilic archaea: a re-evaluation.

Biochemical studies have suggested that, in hyperthermophilic archaea, the metabolic conversion of glucose via the ED (Entner-Doudoroff) pathway generally proceeds via a non-phosphorylative variant. A key enzyme of the non-phosphorylating ED pathway of Sulfolobus solfataricus, KDG (2-keto-3-deoxygluconate) aldolase, has been cloned and characterized previously. In the present study, a comparative genomics analysis is described that reveals conserved ED gene clusters in both Thermoproteus tenax and S. solfataricus. The corresponding ED proteins from both archaea have been expressed in Escherichia coli and their specificity has been identified, revealing: (i) a novel type of gluconate dehydratase (gad gene), (ii) a bifunctional 2-keto-3-deoxy-(6-phospho)-gluconate aldolase (kdgA gene), (iii) a 2-keto-3-deoxygluconate kinase (kdgK gene) and, in S. solfataricus, (iv) a GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase; gapN gene). Extensive in vivo and in vitro enzymatic analyses indicate the operation of both the semi-phosphorylative and the non-phosphorylative ED pathway in T. tenax and S. solfataricus. The existence of this branched ED pathway is yet another example of the versatility and flexibility of the central carbohydrate metabolic pathways in the archaeal domain.

Aldehyde-Lyases↗