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Robert Huber

Publications and source records attributed to Robert Huber.

At least 127 records · Page 7Linked to original sources
PubMed2002

Physiological fIXa activation involves a cooperative conformational rearrangement of the 99-loop.

Coagulation factor IXa (fIXa) plays a central role in the coagulation cascade. Enzymatically, fIXa is characterized by its very low amidolytic activity that is not improved in the presence of cofactor, factor VIIIa (fVIIIa), distinguishing fIXa from all other coagulation factors. Activation of the fIXa-fVIIIa complex requires its macromolecular substrate, factor X (fX). The 99-loop positioned near the active site partly accounts for the poor activity of fIXa because it adopts a conformation that interferes with canonical substrate binding in S2-S4. Here we show that residues Lys-98 and Tyr-99 are critically linked to the amidolytic properties of fIXa. Exchange of Tyr-99 with smaller residues resulted not only in an overall decreased activity but also in impaired binding in S1. Replacement of Lys-98 with smaller and uncharged residues increased activity. Simultaneous mutagenesis of Lys-98, Tyr-177, and Tyr-94 produced an enzyme with 7000-fold increased activity and altered specificity. This triple mutant probably mimics the conformational changes that are physiologically induced by cofactor and substrate binding. It therefore provides a cooperative two-step activation model for fIXa. Tyr-177 locks the 99-loop in an inactive conformation which, in the physiologic complex, is released by cofactor fVIIIa. FX is then able to rearrange the unlocked 99-loop and subsequently binds to the active site cleft.

Amides↗
PubMed2002

The influence of residue 190 in the S1 site of trypsin-like serine proteases on substrate selectivity is universally conserved.

We examined the influence of Ser/Ala190 in the S1 site on P1 substrate selectivity in several serine proteases. The impact of residue 190 on the selectivity was constant, regardless of differences in original selectivity or reactivity. Substrate binding in S1 was optimised in all wild-type enzymes, while the effects on k(cat) depended on the combination of residue 190 and substrate. Mutagenesis of residue 190 did not affect the S2-S4 sites. Pronounced selectivity for arginine residues was coupled with low enzymatic activity, in particular in recombinant factor IXa. This is due to the dominance of the S1-P1 interaction over substrate binding in the S2-S4 sites.

Arginine↗
PubMed2002

Snapshots of the cystine lyase C-DES during catalysis. Studies in solution and in the crystalline state.

The cystine lyase (C-DES) of Synechocystis is a pyridoxal-5'-phosphate-dependent enzyme distantly related to the family of NifS-like proteins. The crystal structure of an N-terminal modified variant has recently been determined. Herein, the reactivity of this enzyme variant was investigated spectroscopically in solution and in the crystalline state to follow the course of the reaction and to determine the catalytic mechanism on a molecular level. Using the stopped-flow technique, the reaction with the preferred substrate cystine was found to follow biphasic kinetics leading to the formation of absorbing species at 338 and 470 nm, attributed to the external aldimine and the alpha-aminoacrylate; the reaction with cysteine also exhibited biphasic behavior but only the external aldimine accumulated. The same reaction intermediates were formed in crystals as seen by polarized absorption microspectrophotometry, thus indicating that C-DES is catalytically competent in the crystalline state. The three-dimensional structure of the catalytically inactive mutant C-DES(K223A) in the presence of cystine showed the formation of an external aldimine species, in which two alternate conformations of the substrate were observed. The combined results allow a catalytic mechanism to be proposed involving interactions between cystine and the active site residues Arg-360, Arg-369, and Trp-251*; these residues reorient during the beta-elimination reaction, leading to the formation of a hydrophobic pocket that stabilizes the enolimine tautomer of the aminoacrylate and the cysteine persulfide product.

Bacterial Proteins↗
PubMed2002

Structures of the tricorn-interacting aminopeptidase F1 with different ligands explain its catalytic mechanism.

F1 is a 33.5 kDa serine peptidase of the alpha/beta-hydrolase family from the archaeon Thermoplasma acidophilum. Subsequent to proteasomal protein degradation, tricorn generates small peptides, which are cleaved by F1 to yield single amino acids. We have solved the crystal structure of F1 with multiwavelength anomalous dispersion (MAD) phasing at 1.8 A resolution. In addition to the conserved catalytic domain, the structure reveals a chiefly alpha-helical domain capping the catalytic triad. Thus, the active site is accessible only through a narrow opening from the protein surface. Two structures with molecules bound to the active serine, including the inhibitor phenylalanyl chloromethylketone, elucidate the N-terminal recognition of substrates and the catalytic activation switch mechanism of F1. The cap domain mainly confers the specificity for hydrophobic side chains by a novel cavity system, which, analogously to the tricorn protease, guides substrates to the buried active site and products away from it. Finally, the structure of F1 suggests a possible functional complex with tricorn that allows efficient processive degradation to free amino acids for cellular recycling.

Amino Acid Sequence↗
PubMed2002

Incorporation of an amide into 5-phosphonoalkyl-6-D-ribitylaminopyrimidinedione lumazine synthase inhibitors results in an unexpected reversal of selectivity for riboflavin synthase vs lumazine synthase.

Several analogues of a hypothetical intermediate in the reaction catalyzed by lumazine synthase were synthesized and tested as inhibitors of both Bacillus subtilis lumazine synthase and Escherichia coli riboflavin synthase. The new compounds were designed by replacement of a two-carbon fragment of several 5-phosphonoalkyl-6-D-ribitylaminopyrimidinedione lumazine synthase inhibitors with an amide linkage that was envisioned as an analogue of a Schiff base moiety of a hypothetical intermediate in the enzyme-catalyzed reaction. The incorporation of the amide group led to an unexpected reversal in selectivity for inhibition of lumazine synthase vs riboflavin synthase. Whereas the parent 5-phosphonoalkyl-6-D-ribitylaminopyrimidinediones were lumazine synthase inhibitors and did not inhibit riboflavin synthase, the amide-containing derivatives inhibited riboflavin synthase and were only very weak or inactive as lumazine synthase inhibitors. Molecular modeling of inhibitor-lumazine synthase complexes did not reveal a structural basis for these unexpected findings. However, molecular modeling of one of the inhibitors with E. coli riboflavin synthase demonstrated that the active site of the enzyme could readily accommodate two ligand molecules.

Amides↗
PubMed2002

Mechanism of the six-electron reduction of nitrite to ammonia by cytochrome c nitrite reductase.

Cytochrome c nitrite reductase catalyzes the six-electron reduction of nitrite to ammonia without the release of potential reaction intermediates, such as NO or hydroxylamine. On the basis of the crystallographic observation of reaction intermediates and of density functional calculations, we present a working hypothesis for the reaction mechanism of this multiheme enzyme which carries a novel lysine-coordinated heme group (Fe-Lys). It is proposed that nitrite reduction starts with a heterolytic cleavage of the N-O bond which is facilitated by a pronounced back-bonding interaction of nitrite coordinated through nitrogen to the reduced (Fe(II)) but not the oxidized (Fe(III)) active site iron. This step leads to the formation of an [FeNO](6) species and a water molecule and is further facilitated by a hydrogen bonding network that induces an electronic asymmetry in the nitrite molecule that weakens one N-O bond and strengthens the other. Subsequently, two rapid one-electron reductions lead to an [FeNO](8) form and, by protonation, to an Fe(II)-HNO adduct. Hereafter, hydroxylamine will be formed by a consecutive two-electron two-proton step which is dehydrated in the final two-electron reduction step to give ammonia and an additional water molecule. A single electron reduction of the active site closes the catalytic cycle.

Ammonia↗
PubMed2002

Salinisphaera shabanensis gen. nov., sp. nov., a novel, moderately halophilic bacterium from the brine-seawater interface of the Shaban Deep, Red Sea.

A novel, moderately halophilic bacterium was isolated from the brine-seawater interface of the Shaban Deep, northern Red Sea. A polyphasic approach was used for the taxonomic characterization of this isolate, with the phenotypic and phylogenetic data clearly showing the distinctiveness of this bacterium. Cells of isolate E1L3A were Gram-negative, monotrichous cocci that showed a remarkable physiological flexibility, as could be seen by the quite broad growth ranges for oxygen, temperature, NaCl, and, to a smaller degree, pH. In addition, it was able to grow from atmospheric pressure up to 15 MPa, making it a piezotolerant bacterium. Phylogenetically, strain E1L3A represents a new, deeply branching lineage within the gamma-Proteobacteria, as determined by 16S rRNA gene sequence analysis. No close relatives are known so far, with sequence similarity to other cultivated members of the gamma-Proteobacteria being lower than 88%. The creation of the new genus Salinisphaera and the new species Salinisphaera shabanensis (DSM 14853; JCM 11575) for this new and highly versatile microorganism is therefore proposed.

Base Composition↗
PubMed2002

Crystal structures of uninhibited factor VIIa link its cofactor and substrate-assisted activation to specific interactions.

Factor VIIa initiates the extrinsic coagulation cascade; this event requires a delicately balanced regulation that is implemented on different levels, including a sophisticated multi-step activation mechanism of factor VII. Its central role in hemostasis and thrombosis makes factor VIIa a key target of pharmaceutical research. We succeeded, for the first time, in recombinantly producing N-terminally truncated factor VII (rf7) in an Escherichia coli expression system by employing an oxidative, in vitro, folding protocol, which depends critically on the presence of ethylene glycol. Activated recombinant factor VIIa (rf7a) was crystallised in the presence of the reversible S1-site inhibitor benzamidine. Comparison of this 1.69A crystal structure with that of an inhibitor-free and sulphate-free, but isomorphous crystal form identified structural details of factor VIIa stimulation. The stabilisation of Asp189-Ser190 by benzamidine and the capping of the intermediate helix by a sulphate ion appear to be sufficient to mimic the disorder-order transition conferred by the cofactor tissue factor (TF) and the substrate factor X. Factor VIIa shares with the homologous factor IXa, but not factor Xa, a bell-shaped activity modulation dependent on ethylene glycol. The ethylene glycol-binding site of rf7a was identified in the vicinity of the 60 loop. Ethylene glycol binding induces a significant conformational rearrangement of the 60 loop. This region serves as a recognition site of the physiologic substrate, factor X, which is common to both factor VIIa and factor IXa. These results provide a mechanistic framework of substrate-assisted catalysis of both factor VIIa and factor IXa.

Binding Sites↗
PubMed2002

Crystal structures of transcription factor NusG in light of its nucleic acid- and protein-binding activities.

Microbial transcription modulator NusG interacts with RNA polymerase and termination factor rho, displaying striking functional homology to eukaryotic Spt5. The protein is also a translational regulator. We have determined crystal structures of Aquifex aeolicus NusG showing a modular design: an N-terminal RNP-like domain, a C-terminal element with a KOW sequence motif and a species-specific immunoglobulin-like fold. The structures reveal bona fide nucleic acid binding sites, and nucleic acid binding activities can be detected for NusG from three organisms and for the KOW element alone. A conserved KOW domain is defined as a new class of nucleic acid binding folds. This module is a close structural homolog of tudor protein-protein interaction motifs. Putative protein binding sites for the RNP and KOW domains can be deduced, which differ from the areas implicated in nucleic acid interactions. The results strongly argue that both protein and nucleic acid contacts are important for NusG's functions and that the factor can act as an adaptor mediating indirect protein-nucleic acid associations.

Amino Acid Sequence↗
PubMed2002

Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase of Methanococcus jannaschii.

The hypothetical protein predicted by the open reading frame MJ0055 of Methanococcus jannaschii was expressed in a recombinant Escherichia coli strain under the control of a synthetic gene optimized for translation in an eubacterial host. The recombinant protein catalyzes the formation of the riboflavin precursor 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 174 nmol mg(-1) min(-1) at 37 degrees C. The homodimeric 51.6-kDa protein requires divalent metal ions, preferentially magnesium, for activity. The reaction involves an intramolecular skeletal rearrangement as shown by (13)C NMR spectroscopy using [U-(13)C(5)]ribulose 5-phosphate as substrate. A cluster of charged amino acid residues comprising arginine 25, glutamates 26 and 28, and aspartates 21 and 30 is essential for catalytic activity. Histidine 164 and glutamate 185 were also shown to be essential for catalytic activity.

Amino Acid Sequence↗
PubMed2002

Enzyme-ligand complexes of pyridoxine 5'-phosphate synthase: implications for substrate binding and catalysis.

Pyridoxine 5'-phosphate (PNP) synthase is the last enzyme in the de novo biosynthesis of vitamin B(6) catalyzing the complicated ring-closure reaction between 1-deoxy-D-xylulose-5-phosphate and 1-amino-acetone-3-phosphate. Here we present the crystal structures of four PNP synthase complexes with substrates and substrate analogs. While the overall fold of the enzyme is conserved in all complexes, characteristic readjustments were observed in the active site. The complementary structural information allowed us to postulate a detailed reaction mechanism. The observed binding mode of substrates indicates how the first reaction intermediate, the Schiff-base conjugate, is formed. The most important mechanistic features are the presence of two phosphate-binding sites with distinct affinities and the existence of a water relay system for the release of reaction water molecules. Furthermore, the complexes provide the basis to rationalize the open-closed transition of a flexible loop located on the C-terminal side of the TIM-barrel. Binding of both substrate molecules to the active site seems to be a prerequisite to trigger this transition. Highly conserved mechanistically important residues in the PNP synthase family imply a similar active site organization and reaction mechanism for all family members. Due to the exclusive presence of PNP synthase in a subset of eubacteria, including several well-known pathogens, and due to its outstanding physiological importance for these organisms, the enzyme appears to be a promising novel target for antibacterial drug design.

Amino Acid Sequence↗
PubMed2002

The 2.0A resolution structure of the catalytic portion of a cyanobacterial membrane-bound manganese superoxide dismutase.

Cyanobacteria are shown to be unique in containing membrane-bound manganese superoxide dismutases (MnSOD). They are homodimeric type 2 membrane proteins that protect this phototrophic organism against oxidative stress. We have determined, for the first time, the 2.0A resolution structure of the catalytic portion of the MnSOD from the filamentous cyanobacterium Anabaena PCC 7120. Within each subunit, both the N-terminal helical hairpin (His94 and His145) and the C-terminal alpha/beta domain (His232 and Asp228) contribute ligands to the catalytic manganese site. Together with a water or hydroxide ion (OH(x)) a five-coordinated trigonal bipyramidal geometry is formed, with OH(x) and His90 forming the axial ligands and manganese shifted out of the equatorial plane in the direction of OH(x). The ligands including OH(x) are tightly constrained by hydrogen bonding with surrounding residues either from the same monomer (Tyr98, Asn144, Trp194, Gln213, Val229, Trp230) or from the neighbouring subunit (Glu231, Tyr235). This underlines the important role of the symmetric dimeric structure of MnSODs in contributing elements to both the active site and the substrate funnel. The Mn cdots, three dots, centered Mn distance (18.4A) is bridged by the hydrogen-bonded His232 of one monomer with Glu231 of the other monomer. A detailed discussion of the structure, a comparison with known structures of soluble MnSODs as well as a model of the cyanobacterial membrane-bound MnSOD is presented.

Amino Acid Sequence↗
PubMed2002

The crystal structure of human alpha1-tryptase reveals a blocked substrate-binding region.

Human mast cell tryptases represent a subfamily of trypsin-like serine proteinases implicated in asthma. Unlike beta-tryptases, alpha-tryptases apparently are proteolytically inactive. We have solved the 2.2A crystal structure of mature human alpha1-tryptase. It reveals a frame-like tetrameric architecture that, surprisingly, does not require heparin-binding for stability. In marked contrast to beta2-tryptase, the Ser214-Gly219 segment, which normally provides the template for substrate binding, is kinked in alpha-tryptase, thereby blocking its non-primed subsites. This so far unobserved subsite distortion is incompatible with productive substrate binding and processing. alpha-Tryptase apparently is trapped in this off-conformation by repulsions and attractions of the Asp216 side-chain. However, proteolytic activity could be generated by an induced-fit mechanism.

Amino Acid Sequence↗
PubMed2002

Formation of metal nanoclusters on specific surface sites of protein molecules.

During vacuum condensation of metals on frozen proteins, nanoclusters are preferentially formed at specific surface sites (decoration). Understanding the nature of metal/protein interaction is of interest for structure analysis and is also important in the fields of biocompatibility and sensor development. Studies on the interaction between metal and distinct areas on the protein which enhance or impede the probability for cluster formation require information on the structural details of the protein's surface underlying the metal clusters. On three enzyme complexes, lumazine synthase from Bacillus subtilis, proteasome from Thermoplasma acidophilum and GTP cyclohydrolase I from Escherichia coli, the decoration sites as determined by electron microscopy (EM) were correlated with their atomic surface structures as obtained by X-ray crystallography. In all three cases, decoration of the same protein results in different cluster distributions for gold and silver. Gold decorates surface areas consisting of polar but uncharged residues and with rough relief whereas silver clusters are preferentially formed on top of protein pores outlined by charged and hydrophilic residues and filled with frozen buffer under the experimental conditions. A common quality of both metals is that they strictly avoid condensation on hydrophobic sites lacking polar and charged residues. The results open ways to analyse the binding mechanism of nanoclusters to small specific sites on the surface of hydrated biomacromolecules by non-microscopic, physical-chemical methods. Understanding the mechanism may lead to advanced decoration techniques resulting in fewer background clusters. This would improve the analysis of single molecules with regard to their symmetries and their orientation in the adsorbed state and in precrystalline assemblies as well as facilitate the detection of point defects in crystals caused by misorientation or by impurities.

Bacillus subtilis↗
PubMed2002

Design, synthesis, and evaluation of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-ribitylaminolumazines as inhibitors of riboflavin synthase and lumazine synthase.

A series of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-D-ribityllumazine were synthesized as inhibitors of both Escherichia coli riboflavin synthase and Bacillus subtilis lumazine synthase. The compounds were designed to bind to both the ribitylpurine binding site and the phosphate binding site of lumazine synthase. In the carboxyalkyl series, maximum activity against both enzymes was observed with the 3'-carboxypropyl compound 22. Lengthening or shortening the chain linking the carboxyl group to the lumazine by one carbon resulted in decreased activity. In the phosphonoxyalkyl series, the 3'-phosphonoxypropyl compound 33 was more potent than the 4'-phosphonoxybutyl derivative 39 against lumazine synthase, but it was less potent against riboflavin synthase. Molecular modeling suggested that the terminal carboxyl group of 6-(3'-carboxypropyl)-7-oxo-8-D-ribityllumazine (22) may bind to the side chains of Arg127 and Lys135 of the enzyme. A hypothetical molecular model was also constructed for the binding of 6-(2'-carboxyethyl)-7-oxolumazine (15) in the active site of E. coli riboflavin synthase, which demonstrated that the active site could readily accommodate two molecules of the inhibitor.

Binding Sites↗
PubMed2002

Structural basis of the adaptive molecular recognition by MMP9.

Matrix metalloproteinase (MMPs) are critical for the degradation of extracellular matrix components and, therefore, need to be regulated tightly. Almost all MMPs share a homologous C-terminal haemopexin-like domain (PEX). Besides its role in macromolecular substrate processing, the PEX domains appear to play a major role in regulating MMP activation, localisation and inhibition. One intriguing property of MMP9 is its competence to bind different proteins, involved in these regulatory processes, with high affinity at an overlapping recognition site on its PEX domain. With the crystal structure of the PEX9 dimer, we present the first example of how PEX domains accomplish these diverse roles. Blade IV of PEX9 mediates the non-covalent and predominantly hydrophobic dimerisation contact. Large shifts of blade III and, in particular, blade IV, accompany the dimerisation, resulting in a remarkably asymmetric homodimeric structure. The asymmetry provides a novel mechanism of adaptive protein recognition, where different proteins (PEX9, PEX1, and TIMP1) can bind with high affinity to PEX9 at an overlapping site. Finally, the structure illustrates how the dimerisation generates new properties on both a physico-chemical and functional level.

Amino Acid Sequence↗
PubMed2002

Functional interactions of HslV (ClpQ) with the ATPase HslU (ClpY).

HslVU is a bacterial homolog of the proteasome, where HslV is the protease that is activated by HslU, an ATPase and chaperone. Structures of singly and doubly capped HslVU particles have been reported, and different binding modes have been observed. Even among HslVU structures with I-domains distal to HslV, no consensus mode of activation has emerged. A feature in the Haemophilus influenzae HslVU structure, insertion of the C termini of HslU into pockets in HslV, was not seen in all other structures of the enzyme. Here we report site-directed mutagenesis, peptide activation, and fluorescence experiments that strongly support the functional relevance of the C terminus insertion mechanism: we find that mutations in HslV that disrupt the interaction with the C termini of HslU invariably lead to inactive enzyme. Conversely, synthetic peptides derived from the C terminus of HslU bind to HslV with 10(-5) M affinity and can functionally replace full HslU particles for both peptide and casein degradation but fail to support degradation of a folded substrate. Thus, the data can be taken as evidence for separate substrate unfoldase and protease stimulation activities in HslU. Enhanced HslV proteolysis could be due to the opening of a gated channel or allosteric activation of the active sites. To distinguish between these possibilities, we have mutated a series of residues that line the entrance channel into the HslV particle. Our mutational and fluorescence experiments demonstrate that allosteric activation of the catalytic sites is required in HslV, but they do not exclude the possibility of channel opening taking place as well. The present data support the conclusion that the H. influenzae structure with I-domains distal to HslV captures the active species and point to significant differences in the activation mechanism of HslV, ClpP, and the proteasome.

ATP-Dependent Proteases↗