Biomedical subjects

Jasper Akerboom

Publications and source records attributed to Jasper Akerboom.

9 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

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

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↗

PubMed2005

Engineering a selectable marker for hyperthermophiles.

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.

Amino Acid Sequence↗

PubMed2004

The structures of inhibitor complexes of Pyrococcus furiosus phosphoglucose isomerase provide insights into substrate binding and catalysis.

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.

Archaeal Proteins↗

PubMed2003

Purification, crystallization and preliminary crystallographic analysis of phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus.

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, b = 42.4, c = 57.3 A, beta = 120.6 degrees and a monomer in the asymmetric unit.

Crystallization↗

PubMed2003

Crystal structure of Pyrococcus furiosus phosphoglucose isomerase. Implications for substrate binding and catalysis.

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.

Amino Acid Sequence↗

PubMed2002

Molecular and biochemical characterization of a distinct type of fructose-1,6-bisphosphatase from Pyrococcus furiosus.

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.

Amino Acid Sequence↗