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Karl Gruber

Publications and source records attributed to Karl Gruber.

At least 19 recordsLinked to original sources
PubMed2007

Stereoselective biocatalytic synthesis of (S)-2-hydroxy-2-methylbutyric acid via substrate engineering by using "thio-disguised" precursors and oxynitrilase catalysis.

3-Tetrahydrothiophenone (4) and 4-phenylthiobutan-2-one (7) were used as masked 2-butanone equivalents to give the corresponding cyanohydrins 5 (79 % yield, 91 % ee) and 8 (95 % yield, 96 % ee) in an enzymatic cyanohydrin reaction applying the hydroxynitrile lyase (HNL) from Hevea brasiliensis. After hydrolysis and desulphurisation the desired intermediate (S)-2-hydroxy-2-methylbutyric acid (10) was obtained with 99 % ee. Interestingly, when applying (R)-selective HNL from Prunus amygdalus again the (S)-cyanohydrin 5 was formed (62 % ee). The absolute configuration of 5 was verified by crystal structure determination of the corresponding hydrolysis derived carboxylate. The fact that both enzymes yield the same enantiomer was analysed and interpreted by molecular modelling calculations.

Aldehyde-Lyases↗
PubMed2006

Serine scanning: a tool to prove the consequences of N-glycosylation of proteins.

N-Glycosylation of proteins is a common posttranslational modification in eukaryotes. Often this results in enhanced protein stability through protection by the attached sugar moieties. Due to its 13 potential N-glycosylation motifs (N-X-T/S), recombinant hydroxynitrile lyase isoenzyme 5 from almonds (PaHNL5) is secreted by the heterologous host Pichia pastoris in a massively glycosylated form, and it shows extraordinary stability at low pH. The importance of N-glycosylation in general, and individual glycosylation sites in particular for stability at low pH were investigated. To identify especially important glycosylation sites asparagine from all N-X-S/T-motifs was replaced by serine. Thus, critical sites, which contributed to overall enzyme activity and/or stability, were identified individually. One glycosylation site revealed to be essential for stability at low pH. After enzymatic deglycosylation, leaving only one acetylglucosamine attached to asparagines, PaHNL5 retained most of its stability at low pH. Protonation effects in the active site as well as higher-order aggregational events upon incubation in low pH were excluded. This study provides evidence for the interconnection of N-glycosylation and stability at low pH for PaHNL5. Moreover, serine scanning was proven to be applicable for quick identification of critical glycosylation sites.

Acetonitriles↗
PubMed2006

Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution.

Esterase EstB from Burkholderia gladioli, showing moderate S-enantioselectivity (E(S)=6.1) in the hydrolytic kinetic resolution of methyl-beta-hydroxyisobutyrate, was subjected to directed evolution in order to reverse its enantioselectivity. After one round of ep-PCR, saturation mutagenesis and high-throughput screening, it was found that different mutations at position 152 (in the vicinity of the active site) increase, decrease and even reverse the natural enantioselectivity of this enzyme. The newly created R-enantioselectivity of the esterase mutein (E(Rapp)=1.5) has been further enhanced by a designed evolution strategy involving random mutations close to the active site. Based on the three-dimensional structure nineteen amino acid residues have been selected as mutation sites for saturation mutagenesis. Mutations at three sites (135, 253 and 351) were found to increase R-enantioselectivity. Successive rounds of saturation mutagenesis at these "hot spots" resulted in an increase in R-enantioselectivity from E(Rapp)=1.5 for the parent mutant to E(Rapp)=28.9 for the best variant which carried four amino acid substitutions. Our results prove designed evolution followed by high-throughput screening to be an efficient strategy for engineering enzyme enantioselectivity.

Binding Sites↗
PubMed2006

Characterization of squalene epoxidase of Saccharomyces cerevisiae by applying terbinafine-sensitive variants.

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G(25)S, D(335)X (W, F, P), and G(210)A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G(30)S and L(37)P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R(269)G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines.

Alleles↗
PubMed2006

Surgical treatment of distal radius fractures with an angle fixed bar palmar plating system: a single center study of 102 patients over a 2-year period.

INTRODUCTION: Most fractures of the distal radius can be treated by conservative means; however, unstable distal radius fractures (DRFs) require surgical fixation. The purpose of this study was to evaluate functional and radiographic results of DRFs treated with a palmar plating system regarding patient's quality of life. METHODS: This study reports a total number of 104 DRFs in 102 patients, operatively treated, over a 2-year period with a mean follow up time of 15.6 +/- 7.2 months The patient's average age was 59.9 +/- 18.0 years. The results were evaluated according to the scores of Gartland and Werley, Castaing and the disabilities of the arm, shoulder, and hand (DASH) outcome questionnaire. RESULTS: Ninety-three patients were evaluated at final examination. Excellent and good results were achieved for 92 (98.9%) patients according to score of Gartland and Werley and for 86 (92.5%) patients regarding the scoring system of Castaing. The DASH outcome questionnaire averaged 8.0 +/- 13.7 points. INTERPRETATION: This study shows, in a representative number of cases, that palmar plating of unstable fractures of the distal radius is a safe and effective treatment modality. Early surgical treatment results in optimal outcome regarding patient's quality of life.

Adolescent↗
PubMed2005

Structure and function of YcnD from Bacillus subtilis, a flavin-containing oxidoreductase.

YcnD from the gram-positive bacterium Bacillus subtilis is a member of a family of bacterial proteins that act as NADH- and/or NADPH-dependent oxidoreductases. Here, we report for the first time on the biochemical characterization of the purified protein, demonstrating that YcnD is an FMN-containing enzyme that can be reduced by NADH or NADPH (Km = 6.4 and 4.4 microM, respectively). In the presence of free FMN as the electron-accepting substrate, the latter reductant showed a ping-pong Bi-Bi reaction mechanism, whereas utilization of NADH is competitively inhibited by this substrate. This finding suggests that NADPH is the physiological reductant of the enzyme. We also show that YcnD reduces nitro-organic compounds, chromate, and a series of azo dyes. The reduction of azo dyes appears to be mediated by free reduced FMN because the reaction is considerably slower in its absence. Structure determination by X-ray crystallography revealed that YcnD folds into a three layer alpha-beta-alpha sandwich strongly resembling the topology of the NADH oxidase superfamily. Similar to homologous bacterial oxidoreductase, YcnD forms homodimers with an extended dimer interface. The biochemical data and the structure are discussed in light of the putative physiological function of YcnD as an oxidoreductase delivering reduced FMN to enzymes that require the reduced cofactor for activity.

Bacillus subtilis↗
PubMed2004

Homocoenzyme B12 and bishomocoenzyme B12: covalent structural mimics for homolyzed, enzyme-bound coenzyme B12.

Efficient electrochemical syntheses of "homocoenzyme B(12)" (2, Co(beta)-(5'-deoxy-5'-adenosyl-methyl)-cob(III)alamin) and "bishomocoenzyme B(12)" (3, Co(beta)-[2-(5'-deoxy-5'-adenosyl)-ethyl]-cob(III)alamin) are reported here. These syntheses have provided crystalline samples of 2 and 3 in 94 and 77 % yield, respectively. In addition, in-depth investigations of the structures of 2 and 3 in solution were carried out and a high-resolution crystal structure of 2 was obtained. The two homologues of coenzyme B(12) (2 and 3) are suggested to function as covalent structural mimics of the hypothetical enzyme-bound "activated" (that is, "stretched" or even homolytically cleaved) states of the B(12) cofactor. From crude molecular models, the crucial distances from the corrin-bound cobalt center to the C5' atom of the (homo)adenosine moieties in 2 and 3 were estimated to be about 3.0 and 4.4 A, respectively. These values are roughly the same as those found in the two "activated" forms of coenzyme B(12) in the crystal structure of glutamate mutase. Indeed, in the crystal structure of 2, the cobalt center was observed to be at a distance of 2.99 A from the C5' atom of the homoadenosine moiety and the latter was found to be present in the unusual syn conformation. In solution, the organometallic moieties of 2 and 3 were shown to be rather flexible and to be considerably more dynamic than the equivalent group in coenzyme B(12). The homoadenosine moiety of 2 was indicated to occur in both the syn and the anti conformations.

Bacterial Proteins↗
PubMed2004

Reaction mechanism of hydroxynitrile lyases of the alpha/beta-hydrolase superfamily: the three-dimensional structure of the transient enzyme-substrate complex certifies the crucial role of LYS236.

The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from Manihot esculenta (MeHNL) are both members of the alpha/beta-hydrolase superfamily. Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for the Hevea enzyme but denied for the Manihot enzyme. We applied a freeze-quench method to prepare crystals of the complex of HbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure. Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme. However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex. Finite difference Poisson-Boltzmann calculations show that in the absence of Lys(236) the catalytic base His(235) would be protonated at neutral pH. All of this suggests that Lys(236) is instrumental for catalysis in several ways, i.e. by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN(-), and by modulating the basicity of the catalytic base. These data complete the elucidation of the reaction mechanism of alpha/beta-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues. Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding.

Acetone↗
PubMed2003

Observation of a short, strong hydrogen bond in the active site of hydroxynitrile lyase from Hevea brasiliensis explains a large pKa shift of the catalytic base induced by the reaction intermediate.

The hydroxynitrile lyase from Hevea brasiliensis (HbHNL) uses a catalytic triad consisting of Ser(80)-His(235)-Asp(207) to enhance the basicity of Ser(80)-O gamma for abstracting a proton from the OH group of the substrate cyanohydrin. Following the observation of a relatively short distance between a carboxyl oxygen of Asp(207) and the N delta(1)(His(235)) in a 1.1 A crystal structure of HbHNL, we here show by (1)H and (15)N-NMR spectroscopy that a short, strong hydrogen bond (SSHB) is formed between the two residues upon binding of the competitive inhibitor thiocyanate to HbHNL: the proton resonance of H-N delta 1(His(235)) moves from 15.41 ppm in the free enzyme to 19.35 ppm in the complex, the largest downfield shift observed so far upon inhibitor binding. Simultaneously, the D/H fractionation factor decreases from 0.98 to 0.35. In the observable pH range, i.e. between pH 4 and 10, no significant changes in chemical shifts (and therefore hydrogen bond strength) were observed for free HbHNL. For the complex with thiocyanate, the 19.35 ppm signal returned to 15.41 ppm at approximately pH 8, which indicates a pK(a) near this value for the H-N epsilon(2)(His(235)). These NMR results were analyzed on the basis of finite difference Poisson-Boltzmann calculations, which yielded the relative free energies of four protonation states of the His(235)-Asp(207) pair in solution as well as in the protein environment with and without bound inhibitor. The calculations explain all the NMR features, i.e. they suggest why a short, strong hydrogen bond is formed upon inhibitor binding and why this short, strong hydrogen bond reverts back to a normal one at approximately pH 8. Importantly, the computations also yield a shift of the free energy of the anionic state relative to the zwitterionic reference state by about 10.6 kcal/mol, equivalent to a shift in the apparent pK(a) of His(235) from 2.5 to 10. This huge inhibitor-induced increase in basicity is a prerequisite for His(235) to act as general base in the HbHNL-catalyzed cyanohydrin reaction.

Aldehyde-Lyases↗
PubMed2003

Data reconciliation, structure analysis and simulation of waste flows: case study Vienna.

The management of complex waste flow systems requires a systematic approach for the handling of data, for obtaining a consistent picture of the system under consideration, and for simulating various policy scenarios and evaluating material control strategies. In this paper the implementation of a useful methodology is presented, which has been developed in previous works and is further enhanced for modelling, identifying, analysing and simulating material flow systems for which at most one measurement per flow is available for a single balancing period. The methodology enables the analyst to cope with missing data and uncertainty in the measurements. A data reconciliation procedure is used to minimise the uncertainty concerning flows by exploiting the redundancies created by restricting the available data to fulfil the available structural information. Statistical tests are introduced to enable the user to check the compatibility of the data with the a priori information. The origins analysis and destination analysis tools allow for a deeper insight into the system structure. Policy scenarios can be treated using the simulation tools. The waste flow system of the city of Vienna has been chosen to demonstrate step-by-step the procedure for building a reliable model and the effective application of the above mentioned methods and tools. Current and future research focuses on models balancing different interrelated quantities simultaneously and on incorporating stock accumulation and depletion behaviour.

Austria↗
PubMed2002

High-throughput protein characterization using mass spectrometric immunoassay.

A high-throughput mass spectrometric immunoassay system for the analysis of proteins directly from plasma is reported. A 96-well format robotic workstation was used to prepare antibody-derivatized affinity pipette tips for subsequent use in the extraction of specific proteins from plasma and deposition onto 96-well format matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) targets. Samples from multiple individuals were screened with regard to the plasma protein transthyretin (TTR), followed by analysis of the same plasma samples for the transthyretin-associated transport protein, retinol-binding protein (RBP). Analyses were able to detect the presence of posttranslationally modified TTR and RBP, as well as a mutation present in the TTR of one individual. Subsequent analyses of wild-type and mutated TTR using enzymatically active MALDI-TOF MS targets were able to identify the site and nature of the point mutation. The approach represents a rapid (approximately 100 samples/2 h, reagent preparation-to-data) and accurate means of characterizing specific proteins present in large numbers of individuals for proteomic and clinical/diagnostic purposes.

Antibody Affinity↗
PubMed2002

Coenzyme B(12) dependent glutamate mutase.

The crystal structure of glutamate mutase with bound coenzyme B(12) suggests a radical shuttling mechanism within the active site of the enzyme. Quantum chemical calculations of the rearrangement in combination with kinetic and mutational studies suggest the catalytic mechanism of this enzyme to proceed via a fragmentation/recombination sequence with intermediates stabilized by partial protonation/deprotonation. Crucial residues in the active site have been identified. Solution structure studies indicate the mechanism of B(12) binding to the apoenzyme.

Binding Sites↗
PubMed2002

The active site of hydroxynitrile lyase from Prunus amygdalus: modeling studies provide new insights into the mechanism of cyanogenesis.

The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction-the enantiospecific formation of alpha-hydroxynitriles--is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C-C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident.

Aldehyde-Lyases↗