Crystallization of Arthrobacter sp. strain 1C N-(1-D-carboxyethyl)-L-norvaline dehydrogenase and its complex with NAD+

The novel NAD+-linked opine dehydrogenase from a soil isolate Arthrobacter sp. strain 1C belongs to an enzyme superfamily whose members exhibit quite diverse substrate specificites. Crystals of this opine dehydrogenase, obtained in the presence or absence of co-factor and substrates, have been shown to diffract to beyond 1.8 ? resolution. X-ray precession photographs have established that the crystals belong to space group P21212, with cell parameters a = 104.9, b = 80.0, c = 45.5 ? and a single subunit in the asymmetric unit. The elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this novel class of dehydrogenases to enable a comparison to be made with other enzyme families and also as the basis for mutagenesis experiments directed towards the production of natural and synthetic opine-type compounds containing two chiral centres.     

Polymer-assisted laser desorption/ionization analysis of small molecular weight compounds

The use of non-polar, small polymers as matrices for the analysis of low molecular weight compounds in polymer-assisted laser desorption/ionization mass spectrometry (PALDI-MS) is demonstrated. The matrices evaluated were either based on an oligothiophene or a benzodioxin backbone. Metallocenes, polycyclic hydrocarbons, a fluorosurfactant, and a subset of small organic compounds with various functionalities, served as model analytes. The mechanism of ionization charge transfer is discussed and ionization potentials for the matrices in the study have been estimated using density functional theory (DFT) calculations. Some of the results are possibly contradictory to the generally accepted limiting conditions for gas-phase charge-transfer reactions. These results are interpreted in the light of energy pooling. Also a new mass calibration procedure for the low-mass region in positive ion mode is presented, and some aspects of the ionization/desorption process leading to radical cations are studied.     

Capillary electrophoretic and mass spectrometric analysis of a polydisperse fluorosurfactant

A fluorosurfactant has been studied using capillary electrophoresis and mass spectrometry. The fluorosurfactant, FC134, can be used as a buffer additive in capillary electrophoresis in order to decrease wall adsorption of proteins and in micellar electrokinetic chromatography. However, it has been discovered that this fluorosurfactant is polydisperse, thus containing substances with different lengths and structures. In this work, the fluorosurfactant sample components were separated by capillary electrophoresis. An uncoated as well as a poly(vinyl alcohol)-coated capillary were used with running electrolytes containing methanol and acetic acid. Following the capillary electrophoretic separation, fractions were collected for further analysis by MALDI-MS. Non-fractionated samples were also analyzed both by MALDI-MS and by ESI-MS.     

Structure-Function Studies with the Mitochondrial Carnitine Palmitoyltransferases I and II

Summary. Mitochondrial carnitine palmitoyltransferases I and II (CPTI and CPTII), together with the carnitine carrier, transport long-chain fatty acyl-CoAs from the cytosol to the mitochondrial matrix for β-oxidation. As an enzyme that catalyzes the rate-limiting step in fatty acid oxidation, CPTI is inhibited by malonyl-CoA, the first intermediate in fatty acid synthesis. Our development of a high level of expression for CPTI and CPTII in P. pastoris, a yeast with no endogenous CPT activity has enabled us to map the malonyl-CoA and substrate binding sites by mutational analysis. Using deletion and substitution mutants of L-CPTI expressed in P. pastoris, we have shown that Glu3 and His5 are necessary for malonyl-CoA inhibition and high-affinity binding of L-CPTI but not for catalysis. Similar studies of M-CPTI clearly establish that the N-terminal residues Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl-CoA inhibition and binding, but not for catalysis. Furthermore, using chimeras between rat and pig L-CPTI, and deletion mutation analysis, we demonstrated that the differences in malonyl-CoA sensitivity observed between the pig and rat L-CPTI were due to differences in the interaction of the first 18 N-terminal amino acid residues with the C-terminal region of the respective enzymes. Consistent with this, the conserved C-terminal residues R601, E603, R606, and K560 were found to be important for L-CPTI activity, malonyl-CoA inhibition and binding, because mutation of these residues decreased malonyl-CoA sensitivity and enzyme activity. We also identified two conserved C-terminal residues in L-CPTI, D567, and E590, that when mutated to alanine cause a substantial increase in malonyl-CoA sensitivity, suggesting a structural basis for the differences in malonyl-CoA sensitivity between L-CPTI and M-CPTI. Our cysteine-scanning mutagenesis of M-CPTI revealed that a single Cys residue, Cys305, was essential for catalysis. In addition, deletion and substitution analysis of the extreme C-terminal region of M-CPTI, suggest that L764 may be important for proper folding and optimal activity. In summary, our structure-function studies with the mitochondrial carnitine palmitoyltransferases I and II have identified critical residues for inhibitor and substrate binding and catalysis.     

Intercalation of Toluidines into α-Zirconium Hydrogenphosphate

Intercalates of o-, m-, and p-toluidine into α-Zr(HPO₄)₂ · H₂O were prepared and characterized by powder X-ray diffraction, thermogravimetric analysis and infrared spectroscopy. As follows from IR, toludine molecules are protonated in the interlayer space. Toluidine molecules are arranged in a bimolecular way in the intercalates containing more than 1.5 toluidine molecules per Zr atom. On the other hand, a monolayer of the toluidine molecules is supposed in the intercalates with less than one toluidine molecule per Zr atom.     

The free energy of a reaction coordinate at multiple constraints: a concise formulation

The free energy as a function of the reaction coordinate (rc) is the key quantity for the computation of equilibrium and kinetic quantities. When it is considered as the potential of mean force, the problem is the calculation of the mean force for given values of the rc. We reinvestigate the PMCF (potential of mean constraint force) method which applies a constraint to the rc to compute the mean force as the mean negative constraint force and a metric tensor correction. The latter allows for the constraint imposed to the rc and possible artefacts due to multiple constraints of other variables which for practical reasons are often used in numerical simulations. Two main results are obtained that are of theoretical and practical interest. First, the correction term is given a very concise and simple shape which facilitates its interpretation and evaluation. Secondly, a theorem describes various rcs and possible combinations with constraints that can be used without introducing any correction to the constraint force. The results facilitate the computation of free energy by molecular dynamics simulations.     

Nanowires for surface enlargement of narrow-bore fused-silica tubing

A method for preparation of silica nanowires with dimensions of d = 10-100 nm, l = 5-500 nm, is described. The nanostructured material is an integral part of the inner surface of narrow bore fused-silica capillary tubing. The wire preparation method is based on a decomposition of 2-chloro-1,1,2-trifluoroethyl methyl ether at elevated temperature and pressure. The silica bulk material is rearranged via a sustained silica-hydrogen fluoride chemistry, and reaction mechanisms for this process are proposed. The method is suitable for preparing long lengths of tubing with the modified surface. It is our belief that the texture of the capillary wall with its increased surface area is useful for applications such as microreactions, catalysis, and high-resolution pressure and/or electrodriven open-tubular liquid chromatography.     

Matrix-assisted and polymer-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of low molecular weight polystyrenes and polyethylene glycols

Recently, matrices based on oligomers of dioxin and thiophene (polymer-assisted laser desorption/ionization (PALDI)) have been described for mass spectrometric (MS) analysis of low molecular weight compounds (Woldegiorgis A, von Kieseritzky F, Dahlstedt E, Hellberg J, Brinck T, Roeraade J. Rapid Commun. Mass Spectrom. 2004; 18: 841-852). In this paper, we report the use of PALDI matrices for low molecular weight polymers. An evaluation with polystyrene and polyethylene glycol showed that no charge transfer ionization occurs. Ionization is mediated through metal ion adduction. Comparison of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) data for two very low molecular weight polymers with data obtained from size-exclusion chromatography (SEC) revealed a systematic difference regarding mean molecular weight and dispersity. Further, the mass spectra obtained with PALDI matrices had a higher signal-to-noise ratio than the spectra obtained with conventional matrices. For polymers with higher molecular weights (>1500 Da), the conventional matrices gave better performance. For evaluation of the MALDI spectra, three non-linear mathematical models were evaluated to model the cumulative distributions of the different oligomers and their maximal values of Mw, Mn and PDI. Models based on sigmoidal or Boltzmann equations proved to be most suitable. Objective modeling tools are necessary to compare different sample and instrumental conditions during method optimization of MALDI analysis of polymers, since the bias between MALDI and SEC data can be misleading.     

Mechanism of decomposition of the human defense factor hypothiocyanite near physiological pH

Relatively little is known about the reaction chemistry of the human defense factor hypothiocyanite (OSCN(-)) and its conjugate acid hypothiocyanous acid (HOSCN), in part because of their instability in aqueous solutions. Herein we report that HOSCN/OSCN(-) can engage in a cascade of pH- and concentration-dependent comproportionation, disproportionation, and hydrolysis reactions that control its stability in water. On the basis of reaction kinetic, spectroscopic, and chromatographic methods, a detailed mechanism is proposed for the decomposition of HOSCN/OSCN(-) in the range of pH 4-7 to eventually give simple inorganic anions including CN(-), OCN(-), SCN(-), SO(3)(2-), and SO(4)(2-). Thiocyanogen ((SCN)(2)) is proposed to be a key intermediate in the hydrolysis; and the facile reaction of (SCN)(2) with OSCN(-) to give NCS(═O)SCN, a previously unknown reactive sulfur species, has been independently investigated. The mechanism of the aqueous decomposition of (SCN)(2) around pH 4 is also reported. The resulting mechanistic models for the decomposition of HOSCN and (SCN)(2) address previous empirical observations, including the facts that the presence of SCN(-) and/or (SCN)(2) decreases the stability of HOSCN/OSCN(-), that radioisotopic labeling provided evidence that under physiological conditions decomposing OSCN(-) is not in equilibrium with (SCN)(2) and SCN(-), and that the hydrolysis of (SCN)(2) near neutral pH does not produce OSCN(-). Accordingly, we demonstrate that, during the human peroxidase-catalyzed oxidation of SCN(-), (SCN)(2) cannot be the precursor of the OSCN(-) that is produced.