Aloysius F. Hartog

The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH.

The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).

A new crosslinker for mass spectrometric analysis of the quaternary structure of protein complexes.

Mass spectrometric structural analysis of crosslinked peptides is a powerful method to elucidate the spatial arrangement of polypeptides in protein complexes. Our aim is to develop bifunctional crosslinkers that, after crosslinking protein complexes followed by proteolytic digestion, give rise to crosslinked peptides that can be readily tracked down by mass spectrometry. To this end we synthesized the crosslinker N-benzyliminodiacetoyloxysuccinimid (BID), which yields stable benzyl cation marker ions upon low-energy collisioninduced dissociation (CID) tandem mass spectrometry. Sensitive detection of the marker ion upon low-energy CID is demonstrated with different BID-crosslinked peptide preparations. With BID it becomes possible to retrieve crosslinked and crosslinker-adducted peptides, without the necessity of purifying crosslinked peptides prior to identification. The basic design of this crosslinker can be varied upon, in order to meet specific crosslinking needs.

CHARACTERIZATION OF THE RESPIRATORY CHAIN FROM CULTURED CRITHIDIA FASCICULATA

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplastids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F0F1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F0F1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F0 subunits (b and c) and all of the F1 subunits. The F0 b subunit has a low degree of similarity to subunit b from other organisms. The F1 alpha subunit is extremely small making the beta subunit the largest F1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.

Probing FtsZ and Tubulin with C8-Substituted GTP Analogs Reveals Differences in Their Nucleotide Binding Sites

The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are widely used in antiproliferative therapy. In this work, we set out to identify selective inhibitors of FtsZ based on the structure of its natural ligand, GTP. We found that GTP analogs with small hydrophobic substituents at C8 of the nucleobase efficiently inhibit FtsZ polymerization, whereas they have an opposite effect on the polymerization of tubulin. The inhibitory activity of the GTP analogs on FtsZ polymerization allowed us to crystallize FtsZ in complex with C8-morpholino-GTP, revealing the binding mode of a GTP derivative containing a nonmodified triphosphate chain.

Laboratory-evolved Vanadium Chloroperoxidase Exhibits 100-Fold Higher Halogenating Activity at Alkaline pH CATALYTIC EFFECTS FROM FIRST AND SECOND COORDINATION SPHERE MUTATIONS

Directed evolution was performed on vanadium chloroperoxidase from the fungus Curvularia inaequalis to increase its brominating activity at a mildly alkaline pH for industrial and synthetic applications and to further understand its mechanism. After successful expression of the enzyme in Escherichia coli, two rounds of screening and selection, saturation mutagenesis of a “hot spot,” and rational recombination, a triple mutant (P395D/L241V/T343A) was obtained that showed a 100-fold increase in activity at pH 8 (kcat = 100 s-1). The increased Km values for Br- (3.1 mm) and H2O2 (16 μm) are smaller than those found for vanadium bromoperoxidases that are reasonably active at this pH. In addition the brominating activity at pH 5 was increased by a factor of 6 (kcat = 575 s-1), and the chlorinating activity at pH 5 was increased by a factor of 2 (kcat = 36 s-1), yielding the “best” vanadium haloperoxidase known thus far. The mutations are in the first and second coordination sphere of the vanadate cofactor, and the catalytic effects suggest that fine tuning of residues Lys-353 and Phe-397, along with addition of negative charge or removal of positive charge near one of the vanadate oxygens, is very important. Lys-353 and Phe-397 were previously assigned to be essential in peroxide activation and halide binding. Analysis of the catalytic parameters of the mutant vanadium bromoperoxidase from the seaweed Ascophyllum nodosum also adds fuel to the discussion regarding factors governing the halide specificity of vanadium haloperoxidases. This study presents the first example of directed evolution of a vanadium enzyme.

An Aptly Positioned Azido Group in the Spacer of a Protein Cross‐Linker for Facile Mapping of Lysines in Close Proximity

Cross‐links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross‐links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross‐linkers that enable isolation of the cleavage products while preserving information about the linkage. We present an amine‐specific cross‐linker, bis(succinimidyl)‐3‐azidomethyl glutarate (BAMG), that fulfils these requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)phosphine (TCEP) in cross‐linked peptides from BAMG‐treated cytochrome c. One pathway leads to cleavage of the cross‐linked species, while in the other the azido group of BAMG is reduced to an amino group without cleavage. Cross‐linked peptides and peptides modified by partially hydrolysed BAMG yield distinct sets of TCEP‐induced reaction products. These can be isolated by reversed‐phase diagonal chromatography and identified by mass spectrometry to reveal the identity of the parent compounds. The ease with which cross‐link‐derived reaction products can be isolated and identified indicates that the mapping of cross‐links in complex biological assemblies and mixtures of protein complexes might become feasible in the near future.

Synthesis of Carbohydrates in a Continuous Flow Reactor by Immobilized Phosphatase and Aldolase

Herein, we report a new flow process with immobilized enzymes to synthesize complex chiral carbohydrate analogues from achiral inexpensive building blocks in a three-step cascade reaction. The first reactor contained immobilized acid phosphatase, which phosphorylated dihydroxyacetone to dihydroxyacetone phosphate using pyrophosphate as the phosphate donor. The second flow reactor contained fructose-1,6-diphosphate aldolase (RAMA, rabbit muscle aldolase) or rhamnulose-1-phosphate aldolase (RhuA from Thermotoga maritima) and acid phosphatase. The immobilized aldolases coupled the formed dihydroxyacetone phosphate to aldehydes, resulting in phosphorylated carbohydrates. A final reactor containing acid phosphatase that dephosphorylated the phosphorylated product yielded the final product. Different aldehydes were used to synthesize carbohydrates on a gram scale. To demonstrate the feasibility of the flow systems, we synthesized 0.6 g of the D-fagomine precursor. By using immobilized aldolase RhuA we were also able to obtain other stereoisomers of the D-fagomine precursor.

Binding and hydrolysis of 2-azido-ATP and 8-azido-ATP by isolated mitochondrial F1: Characterisation of high-affinity binding sites

The kinetic parameters for the hydrolysis by F1 of the photoreactive nucleotide analogue 2-azido-ATP were determined (Vmax, 105 U/mg F1; Km, 250 microM, in the presence of 1.0 mM SO2-3). In the absence of an activating anion, a non-linear relationship in a Lineweaver-Burk plot was found for the hydrolysis of 2-azido-ATP. The 2-azido-analogues of ATP and ADP proved to be good photoaffinity labels causing notable inactivation of the F1-ATPase activity upon irradiation at 360 nm. This inhibition was also used to demonstrate high-affinity binding of these analogues to a catalytic binding site on the F1. High-affinity binding proved to be an Mg2+-requiring process, occurring with both 2-azido-ATP and 2-azido-ADP but hardly or not occurring with 8-azido-AT(D)P. Covalent binding of 2-nitreno-ATP upon irradiation of F1 containing tightly bound [beta-32P]2-azido-ATP results in a proportional inhibition of ATPase activity, extrapolating to 0.92 mol of covalently bound label per mol of F1 needed for the complete inactivation of the enzyme. When the F1 was irradiated in the presence of excess [beta-32P]2-azido-AT(D)P, 3-4 mol of label were bound when the enzyme was fully inactivated. In all cases, all or most of the radioactivity was found on the beta subunits.

Improvement of an acid phosphatase/DHAP-dependent aldolase cascade reaction by using directed evolution

To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild‐type enzyme at pH 6.0. This variant is 17 times more active than the wild‐type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.

Effects of rapamycin and curcumin treatment on the development of epilepsy after electrically induced status epilepticus in rats

Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment strategies in the electrogenic post–status epilepticus (SE) rat model.