Learning about Enzyme Stability against Organic Cosolvents from Structural Insights by Ion Mobility Mass Spectrometry

Ion mobility spectrometry (IMS) coupled with mass spectrometry (MS) enables the investigation of protein folding in solution. Herein, a proof-of-concept for obtaining structural information about the folding of a protein in dependency of the amount of an organic cosolvent in the aqueous medium by means of this IMS-MS method is presented. By analyzing the protein with native nano-electrospray ionization IMS-MS, the impact of acetonitrile as a representative organic cosolvent and/or pH values on the folding of an enzyme was successfully evaluated in a fast and straightforward fashion, as exemplified for an ene reductase from Gluconobacter oxydans. The IMS-MS results are in agreement with findings from the nicotinamide adenine dinucleotide phosphate (NADPH)-based spectrophotometric enzyme activity tests under analogous conditions, and thus, also rationalizing these “wet” analytical data. For this ene reductase, a higher tolerance against CH₃CN in the presence of a buffer was observed by both analytical methods. The results suggest that this IMS-MS methodology could be a useful complementary tool to existing methods in process optimization and fine-tuning of solvent conditions for biotransformations.     

QCR9, the nuclear gene encoding a small subunit of the mitochondrial cytochrome bc1 complex,maps to the right arm of chromosome VII in Saccharomyces cerevisiae

We present here mapping data for QCR9, a nuclear gene encoding a subunit of the ubiquinol-cytochrome c oxidoreductase complex. Deletion of QCR9 results in the inability of cells to grow on non-fermentable carbon sources at 37°C. Thus, qcr9 mutants can be scored by growing cells on YPE/G at 37°C, or followed by the URA3 marker, which was inserted when making the qcr9 deletion strain, JDP1. The location of QCR9 on the right arm of chromosome VII with respect to the previously mapped genes ADE3, SER2 and PET54 is given.     

The effect of weather damage on wheat enzymes

Sound (ungerminated) and germinated wheat kernals (cv Banks) were milled into wholemeal flours, and the activities of a range of enzymes important in baking were determined. Germination of the wheat gave increases in α-amylase (EC 3.2.1.1), protease (azocaseinase), endo-arabinoxylanase (EC 3.2.1.55), endo-carboxymethylcellulase (EC 3.2.1.4) and β-D-glucopyranosidase (EC 3.2.1.21) activities, and a slight increase in activity was detected for β-D-xylopyranosidase (EC 3.2.1.37). β-Amylase (EC 3.2.1.2) glutathione reductase (EC 1.6.2.4) and most of the glycosidase activities tested did not significantly increase on germination.     

Effects of protein and juglone on gypsy moths: Growth performance and detoxification enzyme activity

The individual and interactive effects of dietary protein and juglone on larval performance and midgut detoxification enxymes were investigated for the gypsy moth,Lymantria dispar. The experimental design was a 2 × 3 factorial, with two levels of protein and three levels of juglone. We monitored survival/development rates from egg hatch to pupation and conducted fourth-instar feeding trials for determination of nutritional indices. Enzyme solutions were prepared from midguts of fifth instars and assayed for polysubstrate monooxygenase, esterase, quinone reductase, and glutathione transferase activities. Results showed that low protein levels prolonged development times, increased consumption rates, and reduced pupal weights. Juglone markedly reduced survival, growth, and consumption rates, increased development times, and reduced pupal weights. The interaction between protein and juglone influenced larval digestion efficiencies and female pupal weights. Polysubstrate monooxygenase activities were unaffected by diet, whereas esterase activities increased in response to both low dietary protein and presence of juglone. Low protein levels increased soluble quinone reductase activities but decreased glutathione transferase activities. Glutathione transferase activities were lowest in larvae fed low-protein, high-juglone diets and may have contributed to the especially poor performance of larvae on those diets. Quinone reductase and glutathione transferase are the systems of importance in detoxification of juglone, and moderate to low activities of these enzymes may explain why gypsy moths perform poorly on members of the Juglandaceae.     

Denitrifying kinetics involving the distributed ratio of reductases

A suspended-growth batch reactor was used to denitrify synthetic wastewater containing various proportions of nitrate and nitrite. A competitive phenomenon between nitrate- and nitrite-reductase was studied utilizing various proportions of nitrate and nitrite in an anaerobic environment with a temperature of 30°C and methanol as carbon source. By using a non-linear regression technique, biokinetic constants of the maximum specific reduction rates of nitrate and nitrite (k₁, k₂) and the Monod half-saturation coefficients of nitrate and nitrite (K_s1, K_s2) for the proposed two-step denitrifying kinetics were 1·29 day^?1, 0·89 day^?1 and 14·3 mg NO-N dm^?3, 10.9 mg NO-N dm^?3, respectively. The result obtained from a series of chemostat studies indicated the Monod-type kinetic model was more accurate when the distributed ratio of nitrate- and nitrite-reductase in the proposed two-step denitrifying kinetics was taken into account.     

Effect of Two Wheat Cultivars Differing in Hydroxamic Acid Concentration on Detoxification Metabolism in the AphidSitobion avenae

Hydroxamic acids (Hx) are wheat secondary metabolites conferring resistance for cereals against aphids. The activity of five enzymatic systems were evaluated in the aphid Sitobion avenae reared on the high-Hx wheat cultivar Chagual and the low-Hx wheat cultivar Huayún for 10 generations. Enzyme solutions were prepared from aphid homogenates and assayed for mixed function oxidases (including cytochrome P-450 monooxygenases and NADPH cytochrome c reductase), glutathione S-transferases, esterases, and catalase. Specific activities per aphid individual of cytochrome P-450 monooxygenases, NADPH cytochrome c reductase, glutathione S-transferases, and esterases were significantly increased in wheat cultivars relative to oat (only marginal increase of esterases in Chagual). Aphids fed on cv. Huayún showed an overall higher induction of enzymatic systems than those fed on cv. Chagual. Comparison of these results with reported effects of Hx on detoxifying enzymes in other insects, including aphids, support the hypothesis that these enzymatic pathways play an important role in the detoxification of toxic host-plant secondary metabolites.     

Bacterial Over-Expression and Purification of the 3′phosphoadenosine 5′phosphosulfate (PAPS) Reductase Domain of Human FAD Synthase: Functional Characterization and Homology Modeling

FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3′phosphoadenosine 5′phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble “truncated” form of hFADS lacking the N-terminal domain (Δ_1-328-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ_1-328-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ_1-328-hFADS enzyme shows a slight, but not significant, change of K_m values (0.24 and 6.23 μM for FMN and ATP, respectively) and of k_cat (4.2 × 10⁻² s⁻¹) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ_1-328-hFADS versus wild-type protein.     

Induction of phase II enzyme,quinone reductase,in murine hepatoma cells in vitro by grape extracts and selected phytochemicals

Consumption of fruits and vegetables has been associated with a lowered risk of developing chronic diseases such as cardiovascular disease and cancer. Grapes are rich in phenolics, flavonoids, and resveratrol, which have been suggested to be responsible for the health functions. Thirteen grape varieties and eighteen common phytochemicals were evaluated for their ability to induce mammalian phase II detoxification enzymes – quinone reductase (QR) in Hepa1c1c7 murine hepatoma cells. Amongst all the grape varieties analyzed, Cabernet Franc showed the highest inducible effect on QR with the lowest induction concentration. Quercetin, genistein, and resveratrol exhibited strong QR induction activity amongst the eighteen phytochemicals. The proliferation of Hepa1c1c7 cells was significantly inhibited in a dose-dependent manner after exposure to all of the grape extracts and some of the phytochemicals. The selective index (SI) of grapes and phytochemicals as anticarcinogenic potency was assessed.     

Improved trimethoprim-resistance cassette for prokaryotic selections

Many of the antibiotic resistance elements used in molecular biology have idiosyncratic limitations. For example, beta-lactam selections rely on antibiotics that are unstable to hydrolysis and allow satellite colonies to form upon extended incubation, and tetracycline selections typically give rise to widely varying colony sizes and lower transformation efficiencies. Although prokaryotic Type II dihydrofolate reductase (dfr) genes have long been considered to have potential utility for the selection of plasmids and mobile elements in bacteria, practical limitations to the quality of those selections, mostly relating to background and inefficiency, have led for the most part to their underuse. I describe here the construction of a Type IIa dfr prokaryotic expression cassette that confers strong resistance against trimethoprim (Tmp), a bactericidal dfr inhibiting antibiotic. The Tmp-resistance cassette provides consistent and efficient selections and plasmid transformation frequencies equivalent to those encountered with beta-lactamases.     

Insights into the structure–function relationship of disease resistance protein HCTR in maize (Zea mays L.): A computational structural biology approach

The disease resistance gene Hm1 of maize encodes a NADPH-dependent reductase enzyme, HC-toxin reductase (HCTR) that detoxifies the HC toxin secreted by the race specific fungus Cochliobolus carbonum race 1. HCTR enzyme shares 29.6% sequence identity with dihydroflavonol reductase (DFR) of grape, a key enzyme involved in flavonoid biosynthesis. Here we report the comparative modelling, molecular dynamics simulation and docking studies to explain the structure–function relationship and the mode of cofactor (NADPH) binding in HCTR enzyme at the molecular level. The nucleotide binding domain of modelled HCTR adopts a classic Rossmann fold and possesses a consensus glycine rich GxGxxG motif. Molecular simulation studies suggested that HCTR model retained stability throughout the simulation in aqueous solution. HCTR model showed considerable structural identities with the cofactor binding site of DFR, but significant difference in the catalytic site might be the reason of functional divergence between these families of proteins. Similarly electrostatic surface potential analysis of both HCTR and DFR revealed profound variations in the charge distribution over the substrate binding site, which can be correlated with the sequence variability and may suggest distinct substrate-binding patterns and differences in the catalytic mechanism. Docking results indicated Phe19, Gly21, Arg40, Thr90, Gly208, Arg218, Glu221 and Thr222 are important residues for cofactor (NADPH) binding through strong hydrogen bonding and electrostatic interactions. Alanine scanning and analysis of docking energies of mutant proteins suggested that Phe19, and Arg40 are two critical residues for the cofactor binding. The result from the present study is expected to pave the way for exploration of similar genes in other economically important crop varieties.