Aldose reductase and the importance of experimental design

Kinetic studies on the AR (aldose reductase) protein have shown that it does not behave as a classical enzyme in relation to ring aldose sugars. As with non-enzymatic glycation reactions, there is probably a free radical element involved derived from monosaccharide autoxidation. in the case of AR, there is free radical oxidation of NADPH by autoxidizing monosaccharides, which is enhanced in the presence of the NADPH-binding protein. Thus any assay for AR based on the oxidation of NADPH in the presence of autoxidizing monosaccharides is invalid, and tissue AR measurements based on this method are also invalid, and should be reassessed. AR exhibits broad specificity for both hydrophilic and hydrophobic aldehydes that suggests that the protein may be involved in detoxification. The last thing we would want to do is to inhibit it. ARIs (AR inhibitors) have a number of actions in the cell which are not specific, and which do not involve them binding to AR. These include peroxy-radical scavenging and effects of metal ion chelation. The AR/ARI story emphasizes the importance of correct experimental design in all biocatalytic experiments. Developing the use of Bayesian utility functions, we have used a systematic method to identify the optimum experimental designs for a number of kinetic model data sets. This has led to the identification of trends between kinetic model types, sets of design rules and the key conclusion that such designs should be based on some prior knowledge of K-m and/or the kinetic model. We suggest an optimal and iterative method for selecting features of the design such as the substrate range, number of measurements and choice of intermediate points. The final design collects data suitable for accurate modelling and analysis and minimizes the error in the parameters estimated, and is suitable for simple or complex steady-state models.

Preliminary X-ray diffraction analysis of YqjH from Escherichia coli: a putative cytoplasmic ferri-siderophore reductase

YqjH is a cytoplasmic FAD-containing protein from Escherichia coli; based on homology to ViuB of Vibrio cholerae, it potentially acts as a ferri-siderophore reductase. This work describes its overexpression, purification, crystallization and structure solution at 3.0 A resolution. YqjH shares high sequence similarity with a number of known siderophore-interacting proteins and its structure was solved by molecular replacement using the siderophore-interacting protein from Shewanella putrefaciens as the search model. The YqjH structure resembles those of other members of the NAD(P)H:flavin oxidoreductase superfamily.

Epoxide ring opening in a zinc(II) complex in water without any Lewis acid catalyst: formation of only one diastereomer out of 2

The epoxide ring in 5,6-dihydro-5,6-epoxy-1,10-phenanthroline (L) opens up in its reaction with 4-methylaniline and 4-methoxyaniline in water in equimolar proportion at room temperature without any Lewis acid catalyst to give a monohydrate of 6-(4-methyl-phenylamino)-5,6-dihydro-1,10-phenanthrolin-5-ol (L′·H2O) and 6-(4-methoxyphenyl-amino)-5,6-dihydro-1,10-phenanthrolin-5-ol (L″) respectively. Reaction time decreases from 72 to 14 h in boiling water. But the yields become less. Reaction of L with Zn(ClO4)2·6H2O in methanol in 3:1 molar ratio at room temperature affords white [ZnL3](ClO4)2·H2O. The X-ray crystal structure of the acetonitrile solvate [ZnL3](ClO4)2·MeCN has been determined which shows that the metal has a distorted octahedral N6 coordination sphere. [ZnL3](ClO4)2·2H2O reacts with 4-methylaniline and 4-methoxyaniline in boiling water in 1:3 molar proportion in the absence of any Lewis acid catalyst to produce [ZnL′3](ClO4)2·4H2O and [ZnL″3](ClO4)2·H2O, respectively in 1–4 h time in somewhat low yield. In the 1H NMR spectra of [ZnL′3](ClO4)2·4H2O and [ZnL″3](ClO4)2·H2O, only one sharp methyl signal is observed implicating that only one diastereomer out of the 23 possibilities is formed. The same diastereomers are obtained when L′·H2O and L″ are reacted directly with Zn(ClO4)2·6H2O in tetrahydrofuran at room temperature in very good yields. Reactions of L′·H2O and L″ with Ru(phen)2Cl2·2H2O (phen = 1,10-phenanthroline) in equimolar proportion in methanol–water mixture under refluxing condition lead to the isolation of two diastereomers of [Ru(phen)2L′](ClO4)2·2H2O and [Ru(phen)2L″](ClO4)2·2H2O.