Crystallization and preliminary X-ray diffraction studies of mandelonitrile lyase from almonds

Single crystals of three different isoenzymes of (R)?(+) mandelonitrile lyase (hydroxynitrile lyase) from almonds (Prunus amygdalus) have been obtained by hanging drop vapor diffusion using polyethylene glycol 4000 and isopropanol as co-precipitants. The crystals belong to the monoclinic space group P2_l with unit cell parameters a = 69.9, b = 95.1, c = 95.6 Å, and β = 118.5°. A complete set of diffraction data has been collected to 2.6 Å resolution on native crystals of isoenzyme III. © 1994 Wiley-Liss, Inc.     

Crystal structure of hydroxynitrile lyase from Sorghum bicolor in complex with the inhibitor benzoic acid: a novel cyanogenic enzyme

The crystal structure of the hydroxynitrile lyase from Sorghum bicolor (SbHNL) in complex with the inhibitor benzoic acid has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 16.5%. The SbHNL sequence places the enzyme in the alpha/beta hydrolase family where the active site nucleophile is predicted to be organized in a characteristic pentapeptide motif which is part of the active site strand-turn-helix motif. In SbHNL, however, a unique two-amino acid deletion is next to the putative active site Ser158, removing thereby the putative oxyanion hole-forming Tyr residue. The presented X-ray structure shows that the overall folding pattern of SbHNL is similar to that of the closely related wheat serine carboxypeptidase (CPD-WII); however, the deletion in SbHNL is forcing the putative active site residues away from the expected hydrolase binding site toward a small hydrophobic cleft, which also contains the inhibitor benzoic acid, defining thereby a completely different SbHNL active site architecture where the traditional view of a classic triad is not given any more. Rather, we propose a mechanism involving general base catalysis by the carboxy-terminal Trp270 carboxyl group and proton transfer toward the leaving nitrile group by an active site water molecule. The unexpected interactions of the inhibitor with the new SbHNL active site also reveal the structural basis for the enzyme's limited substrate specificity. The implications of this structure on the evolution of catalysis in the hydroxynitrile lyase superfamily are discussed.     

Experimental Investigation of the Cd-Pr Phase Diagram

The complete Cd-Pr equilibrium phase diagram was investigated with a combination of powder-XRD, SEM and DTA. All intermetallic compounds within this system, already reported in literature, could be confirmed: CdPr, Cd₂Pr, Cd₃Pr, Cd₄₅Pr₁₁, Cd₅₈Pr₁₃, Cd₆Pr and Cd₁₁Pr. The corresponding phase boundaries were determined at distinct temperatures. The homogeneity range of the high-temperature allotropic modification of Pr could be determined precisely and a limited solubility of 22.1 at.% Cd was derived. Additionally, single-crystal X-ray diffraction was employed to investigate structural details of Cd₂Pr; it is isotypic to the AlB₂-type structure with a z value of the Cd site of 0.5. DTA results of alloys located in the adjacent two-phase fields of Cd₂Pr suggested a phase transformation between 893 and 930°C. For the phase Cd₃Pr it was found that the lattice parameter a changes linearly with increasing Cd content, following Vegard’s rule. The corresponding defect mechanism could be evaluated from structural data collected with single-crystal XRD. Introduction of a significant amount of vacancies on the Pr site and the reduction in symmetry of one Cd position (8c to 32f) resulted in a noticeable decrease of all R-values.     

Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana.

The nitrilase AtNIT1 from Arabidopsis thaliana was overexpressed in Escherichia coli with an N-terminal His6 tag and purified by zinc chelate affinity chromatography in a single step almost to homogeneity in a 68% yield with a specific activity of 34.1 U.mg-1. The native enzyme (approximately 450 kDa) consists of 11-13 subunits (38 kDa). The temperature optimum was determined to be 35 degrees C and a pH optimum of 9 was found. Thus, recombinant AtNIT1 resembles in its properties the native enzyme and the nitrilase from Brassica napus. The stability of AtNIT1 could be significantly improved by the addition of dithiothreitol and EDTA. The substrate range of AtNIT1 differs considerably from those of bacterial nitrilases. Aliphatic nitriles are the most effective substrates, showing increasing rates of hydrolysis with increasing size of the residues, as demonstrated in the series butyronitrile, octanenitrile, phenylpropionitrile. In comparison with 3-indolylacetonitrile, the rate of hydrolysis of 3-phenylpropionitrile is increased by a factor of 330, and the Km value is reduced by a factor of 23. With the exception of fluoro, substituents in the alpha position to the nitrile function completely inhibit the hydrolysis.     

Kinetic Analysis and Simulation Studies for Lipase-Catalysed Resolution of Racemic 2-Methyl-1-Pentanol

The lipase-catalysed optical resolution of a racemic mixture of 2-methyl-l-pentanol by transesterifica-tion using vinyl acetate as acyl donor has been studied experimentally. A mechanistic model has been developed for the double-substrate reaction sequence treating both enantiomers as competing substrates. The model is based upon a ping-pong mechanism with alternative substrates involving an acyl-enzyme intermediate. The kinetic constants of the model have been evaluated using initial rate experiments and nonlinear regression analysis. The model successfully predicts the evolution of the enantiomeric excess of substrate (ee_R) and the degree of conversion with time for batch experiments with various initial concentrations of vinyl acetate and (R,S)-2-methyl-1-pentanol. Furthermore, the rate equations have been used to theoretically study the dynamic progression of a continuous enzyme-catalysed resolution process. The enantiomeric excess as a function of conversion for different process configurations is discussed. It is found, that the maximum attainable ee_R is strongly dependent on the residence time distribution of the continuous reactor and is rather low for a continuous stirred tank reactor (CSTR) due to competitive inhibition effects.     

Anatomically adapted, HA coated SBG stem--ten years of successful implantation.

For stable implantation of anatomically conforming femur prostheses, modifications that accommodate curving and torsion are necessary. In accordance to this concept, the anatomically shaped SBG stem has been developed. The first consecutive implantations were evaluated. 194 primary arthroplasties with uncemented anatomical, HA coated SBG stems were performed. Mean patient age was 61 years. 143 patients with 151 (78%) SBG stems were followed-up clinically and radiologically after an average of 10 (9-11) years. So far, only one implant had to be revised. Survivorship with revision of the femoral component is 99.5% at ten years. The mean postoperative Harris Hip Score was 92. Patients over 75 years had a score of 89; younger patients under 40 years scored 94. Sclerotic lines were seen in zone I or VII in 8%. Minor femoral remodeling (Type 1) occurred proximally in 75%. Minor thickening of the femur at the level of the tip of the prosthesis could be observed in 27% of the cases. There is no correlation between compacta thickening and clinical symptoms (p=0.15). The anatomical shape, the oval diameter and the longitudinal grooves secure stable primary fixation. Together with the HA coating, which enhances osseointegration, the SBG stem has a high success rate in the medium and long term.