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.     

Combining multiset resolution and segmentation for hyperspectral image analysis of biological tissues

Hyperspectral images can provide useful biochemical information about tissue samples. Often, Fourier transform infrared (FTIR) images have been used to distinguish different tissue elements and changes caused by pathological causes. The spectral variation between tissue types and pathological states is very small and multivariate analysis methods are required to describe adequately these subtle changes.     

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.     

Catalytic antibodies for complex reactions

Success in generating catalytic antibodies as enzyme mimics lies in the strategic design of the transition-state analog (TSA) for the reaction of interest, and careful development of screening processes for the selection of antibodies that are catalysts. Typically, the choice of TSA structure is straightforward, and the criterion for selection in screening is often binding of the TSA to the antibody in a microtiter-plate assay. This article emphasizes the problems of TSA design in complex reactions and the importance of selecting antibodies on the basis of catalysis as well as binding to the TSA. The target reaction is the derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanideion. The desired outcome is selective catalysis of formation of the fluorescent derivative in preference to nonfluorescent side-products. In the study, TSA design was directed toward the reaction branch leading to the fluorescent product. Here, we describe a microtiter plate-based assay that is capable of detecting antibodies showing catalytic activity atan early stage. Of the antibodies selected, 36% showed no appreciable binding to any of the substrates tested, but did show catalytic activity in deriving one or more of the amino acids screened. In contrast, only two out of 77 clones that showed binding did not show catalysis. Thus, in this complex system, observation of binding is a good predictor of the presence of catalytic activity, and failure to observe binding is a poor predictor of the absence of catalytic activity.     

Catalytic antibodies for complex reactions: hapten design and the importance of screening for catalysis in the generation of catalytic antibodies for the NDA/CN reaction

Success in generating catalytic antibodies as enzyme mimics lies in the strategic design of the transition-state analog (TSA) for the reaction of interest, and careful development of screening processes for the selection of antibodies that are catalysts. Typically, the choice of TSA structure is straightforward, and the criterion for selection in screening is often binding of the TSA to the antibody in a microtiter-plate assay. This article emphasizes the problems of TSA design in complex reactions and the importance of selecting antibodies on the basis of catalysis as well as binding to the TSA. The target reaction is the derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide ion. The desired outcome is selective catalysis of formation of the fluorescent derivative in preference to nonfluorescent side-products. In the study, TSA design was directed toward the reaction branch leading to the fluorescent product. Here, we describe a microtiter plate-based assay that is capable of detecting antibodies showing catalytic activity at an early stage. Of the antibodies selected, 36% showed no appreciable binding to any of the substrates tested, but did show catalytic activity in derivatizing one or more of the amino acids screened. In contrast, only two out of 77 clones that showed binding did not show catalysis. Thus, in this complex system, observation of binding is a good predictor of the presence of catalytic activity, and failure to observe binding is a poor predictor of the absence of catalytic activity.