Cytochrome P450s: creating novel ligand sets

Cytochromes P450 are a ubiquitous group of hemoproteins that perform vital cellular reactions in all lifeforms. Until recently, it was thought that P450s contained non-covalently bound heme. However, it was established that covalent linkage of the heme macrocycle occurs naturally in one major group of the P450 superfamily. The reaction involves heme linkage to a conserved amino acid and is autocatalytic, occurring as a consequence of P450 turnover. This finding presents opportunities to engineer biotechnologically important P450s to covalently link the heme, in order to stabilize cofactor binding and to enhance operational stability of these P450s. This opportunity has been taken in studies on two important bacterial P450s and has produced variants with intriguingly different properties. In this article we survey the developments in the field, the relationships with heme macrocycle ligations in other proteins and the important impact that recent studies of heme ligation have had on our general appreciation of P450 structure and mechanism.     

Approach to the synthesis of cladiell-11-ene-3,6,7-triol

[reaction: see text] The synthesis of an advanced intermediate in the synthesis of the title compound has been achieved. Key steps include an Ireland-Claisen rearrangement to install the C7 tertiary alcohol stereocenter, an SN2' reaction of an alkoxymethyl Cu reagent, and a diastereoselective Re-catalyzed allylic alcohol transposition.     

Characterisation of legumes by enzymatic hydrolysis,microdialysis sampling,and micro-high-performance anion-exchange chromatography with electrospray ionisation mass spectrometry

An assay based on enzymatic hydrolysisand microdialysis sampling, micro-high-performance anion-exchange chromatography (micro-HPAEC) with electrospray ionisation mass spectrometry (ESI-MS) for the characterisation of legumes is presented. Characterisation of two bean varieties; Phaseolus mungo and P. acutifilous was based upon enzymatic hydrolysis using an endo-beta-mannanase from Aspergillus niger with subsequent analysis of the hydrolysates with HPAEC-MS. The hydrolysates were detected in the positive ionisation mode after desalting the chromatographic effluent, employing a cation-exchange membrane desalting device with water as the regenerating liquid. Mass chromatograms, acquiredafter hydrolysis of both bean samples for 12 h, showed two different profiles of hydrolysates. The P. mungo bean hydrolysate showed the presence of saccharides with a degree of polymerisation (DP) in the range of 2-6, whereas that of P. acutifilous showed only DPs of 2-5. Both bean samples had one type of DP 2, but showed different types of DPs 3, 4 and 5. Only the P. mungo sample showed the presence of DP 6. The most abundant fraction for P. mungo was DP 4, whereas that for P. acutifilous was DP 5. Tandem MS of the hydrolysates showed that the DP 2 hydrolysates observed for the samples were of the same type, having a 1,6 linkage. Also tandem MS data for DPs 3, 4, and 5 showed that similar hydrolysates were present within the same sample as well as among the two samples. The data also showed the existence of 1,6 linkages for DP 3, 4, and 5 hydrolysates. The single enzymatic hydrolysis in combination with microdialysis and HPAEC with ESI-MS proved to be sufficient and reproducible for profiling and showing the difference between the two bean samples.     

Thermodynamics of the As(III)-thiol interaction: arsenite and monomethylarsenite complexes with glutathione, dihydrolipoic acid, and other thiol ligands

Colorimetric (near-UV absorption spectroscopy) and calorimetric (isothermal titration calorimetry) methods have been used to quantify the equilibrium and thermodynamics of arsenite and monomethylarsenite (MMA) coordinating to glutathione (GSH) and the dithiols dimercaptosuccinic acid (DMSA), dihydrolipoic acid (DHLA), and dithiothreitol (DTT). We found that both arsenite and MMA form moderately stable complexes (beta = 10(6)-10(7)) with GSH; that arsenite forms a particularly stable 2:3 complex (beta approximately 10(18)) with the biological cofactor DHLA; that MMA has a somewhat higher affinity than arsenite for thiol ligands; and that entropic factors modulate the overall stability of As(III) complexes with thiols, which are favored by the exothermic formation of As(III)-thiolate bonds. The implications of these results for arsenic toxicity are discussed.     

Fitting differential equation models to observed economic data—I. quasilinearization

This paper discusses the fitting of differential equation models to economic data. In particular, it treats the problem of describing the growth of capital in terms of a differential equation containing several parameters. The parameters are to be estimated on the basis of data. This estimation problem is formulated as a nonlinear boundary value problem. The rapidly convergent successive approximation method of quasilinearization is described and applied. Representative results of numerical experiments are presented, showing the effectiveness of the approach. Suggestions for additional studies are made.