Evolution of protein function can be driven by positive selection of advantageous nonsynonymous codon mutations that arise following gene duplication. By observing the presence and degree of site-specific positive selection for change between divergent paralogs, residue positions responsible for functional changes can be identified. We applied this analysis to genes encoding Mu class glutathione transferases, which differ widely in substrate specificities. Approximately 3% of the amino acid residue positions, both near to and distant from the active site, are under statistically significant positive selection for change. Relevant human glutathione transferase (GST) M1-1 and GST M2-2 codons were mutated. A chemically conservative threonine to serine mutation in GST M2-2 elicited a 1,000-fold increase in specific activity with the GST M1-1-specific substrate trans-stilbene oxide and a 30-fold increase with the alternative epoxide substrates styrene oxide and nitrophenyl glycidol. The reverse mutation in GST M1-1 resulted in reciprocal decreases in activity. Thus, identification of hypervariable codon positions can be a powerful aid in the redesign of protein function, lessening the requirement for extensive mutagenesis or structural knowledge and sometimes suggesting mutations that would otherwise be considered functionally conservative.

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hGSTA3-3 (human Alpha-class glutathione transferase 3-3) efficiently catalyses steroid Delta(5)-Delta(4) double-bond isomerization in vitro, using glutathione as a cofactor. This chemical transformation is an obligatory reaction in the biosynthesis of steroid hormones and follows the oxidation of 3beta-hydroxysteroids catalysed by 3beta-HSD (3beta-hydroxysteroid dehydrogenase). The isomerization has commonly been ascribed to a supplementary function of 3beta-HSD. The present study is the first to provide evidence that hGSTA3-3 contributes to this step in steroid hormone biosynthesis in complex cellular systems. First, we find glutathione-dependent Delta(5)-Delta(4) isomerase activity in whole-cell extracts prepared from human steroidogenic cells. Secondly, effective inhibitors of hGSTA3-3 dramatically decrease the conversion of Delta(5)-androstene-3,17-dione into Delta(4)-androstene-3,17-dione in cell lysates. Thirdly, we show that RNAi (RNA interference) targeting hGSTA3-3 expression decreases by 30% the forskolin-stimulated production of the steroid hormone progesterone in a human placental cell line. This effect is achieved at low concentrations of two small interfering RNAs directed against distinct regions of hGSTA3-3 mRNA, and is weaker in unstimulated cells, in which hGSTA3-3 expression is low. The results concordantly show that hGSTA3-3 makes a significant contribution to the double-bond isomerization necessary for steroid hormone biosynthesis and thereby complements the indispensable 3beta-hydroxysteroid oxidoreductase activity of 3beta-HSD. The results indicate that the lower isomerase activity of 3beta-HSD is insufficient for maximal rate of cellular sex hormone production and identify hGSTA3-3 as a possible target for pharmaceutical intervention in steroid hormone-dependent diseases.

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The biosynthesis of small molecules can be fine-tuned by (re)engineering metabolic flux within cells. We have adapted this approach to optimize an in vivo selection system for the conversion of prephenate to phenylpyruvate, a key step in the production of the essential aromatic amino acid phenylalanine. Careful control of prephenate concentration in a bacterial host lacking prephenate dehydratase, achieved through provision of a regulable enzyme that diverts it down a parallel biosynthetic pathway, provides the means to systematically increase selection pressure on replacements of the missing catalyst. Successful differentiation of dehydratases whose activities vary over a >50,000-fold range and the isolation of mechanistically informative prephenate dehydratase variants from large protein libraries illustrate the potential of the engineered selection strain for characterizing and evolving enzymes. Our approach complements other common methods for adjusting selection pressure and should be generally applicable to any selection system that is based on the conversion of an endogenous metabolite. directed evolution ͉ enzyme design ͉ genetic complementation ͉ prephenate dehydratase ͉ tetracycline promoter

Abolghasem (2019) A single-shot diagnostic platform based on copper nanoclusters coated with cetyl trimethylammonium bromide for determination of carbamazepine in exhaled breath condensate. Microchimica Acta, 186 (3). 194 1-8.ABSTRACT 31 A fluorescent nanoprobe is designed for the determination of carbamazepine (CBZ) in exhaled 32 breath condensate (EBC) of patients receiving CBZ. The probe consists of copper nanoclusters 33 (Cu NCs) coated with cetyl trimethylammonium bromide. The interaction of probe with CBZ 34 results in blocking non-radiative e -/h + recombination defect sites on the surface of Cu NCs and 35 consequently enhancing the fluorescence intensity of Cu NCs. The experimental conditions were 36 optimized by using a response surface methodology (central composite design). Under the 37 optimized conditions, the calibration plot is linear in the 0.2 to 20 µg mL -1 CBZ concentration 38 range (excitation/emission wavelength: 290/480 nm) and the detection limit is as low as 0.08 µg 39 mL -1 . The intra-day and inter-day relative standard deviations for six replicated measurements of 40 10 µg mL -1 CBZ are 3.9 % and 4.8 %, respectively. The method was applied for the 41 determination of CBZ level in EBC of patients receiving CBZ. The accuracy of the method was 42 confirmed by HPLC-UV analysis as a references method. 43 44 45 surface methodology; central composite design; 47 48 3 49 1. Introduction 50 Carbamazepine (5H-dibenzo [b,f] azepine-5-carboxamide, CBZ) is a carboxamide 51 derivative of iminostilbene [1] and widely used for the therapy of patients with partial and 52 generalized tonic-clonic epilepsy and psychiatric diseases [2]. So far, various methods, such as 53 liquid chromatography-electrospray ionization mass spectrometry method (LC-EIA-MS) in 54 serum samples [3], LC-MS-MS method in whole blood, serum and plasma [4], ion mobility 55 spectrometry in formulation samples [5], stacking capillary electrophoresis method in serum 56 samples [6], high-performance liquid chromatography-diode array detection assay after a simple 57 sample preparation method by a packed sorbent in plasma samples [7], online solid phase 58 extraction -LC coupled with high resolution mass spectrum under targeted MS/MS analysis 59 mode in serum sample [8], and a gas chromatography method [9] have been developed for the 60 determination of CBZ. Although the advantages of such methods are undeniable, most of these 61 techniques need a sample preparation procedure to remove matrix components or a derivatization 62 step before analysis and also a labelled internal standard. So, development of an easy-to-use 63 method that can be applied for off-and on-line determination of drugs concentrations in 64 biological samples has always been of interest in bioanalytical and clinical applications. It is 65 important to note that the assay of biomolecules should be fast, cost-effective, selective, and 66 sensitive. For this purpose, increasing attention has been focused on the construction of 67 fluorescent nanoprobes for drugs [10]. Qua...

Glutathione transferases (GSTs) play a central role in the cellular defense against harmful endogenous compounds and xenobiotics in mouse and man. The gastrointestinal channel is constantly exposed to bacteria, bacterial products, and xenobiotics. In the present study the distribution of alpha, mu, and pi class GSTs was examined immunohistologically in the colon of conventional and germ-free (GF) mice subjected to experimental colitis. The tissues samples were from conventional mice with and without colitis induced by dextran sulfate sodium (DSS); GF mice treated with DSS or carrageenan; and GF mice inoculated with normal mouse bacterial flora as well as with Lactobacillus GG. In conventional as well as in GF mice the mu and pi class GSTs showed reduced intestinal expression when colitis was induced. In con-rast, the level of GSTs reacting with antibodies directed against the alpha class, in particular mGST A4-4, was elevated after induction of inflammation. Of special interest is mGST A4-4 because of its high catalytic activity with toxic products of lipid peroxidation. In the colon of conventionalized GF mice that were given mouse intestinal flora, the mGST A4-4 expression was increased with time for several weeks, but then showed a decrease to a normal level. Additionally, the inoculation of GF mice with Lactobacillus GG induced all the intestinal GSTs studied.