No description available.

No description available.

Human methionine synthase reductase (MSR) is a 78-kDa diflavin enzyme involved in folate and methionine metabolism. It regenerates the cofactor of methionine synthase (MS), cob(II), to reduce inactive MS. MSR and one of its the FAD/NADPH binding domain were cloned as GST-tagged fusion proteins for expression and purification in Escherichia coli. And a 1.9 Å Crystals of the FAD/NADPH binding domain of MSR with and without NADP+ were produced and carried out X-ray diffraction experiment and the structure of the crystal was solved by molecule replacement method. The activation domain of human MS was also expressed and purified in Escherichia coli and crystallization conditions determined. A new expression vector for full-length MSR, which contains a N-terminal GST tag, and C-terminal 6× His tag, was constructed and validated by sequencing, restriction enzymes digestion and successfully expressed in E. coli and Yeast Pichia pastoris. Based on the structure information, site-directed mutagenesis on the two positions Asp652 and trpytophan697 of MSR were designed and completed. The variants D652A, D652R, D652N of the FAD/NADPH binding domain of MSR and the variants D652A,D652R,D652N, W696A,W697H of the full-length MSR were cloned and expressed in BL21 (E. coli). The proteins of these mutants were purified by affinity chromatography, anion exchange chromatography and gel filtration chromatography. And the kinetic studies on these variants of MSR were investigated in steady state kinetic study, steady state inhibition studies, stopped-flow pre steady-state kinetic and redox potential studies. Compared with the data of the wild type MSR, the turnover number of variants all decreased, the catalytic ability become lower and the midpoint potential of cofactor FAD occurred positive shift. Both 2'5-ADP and NADP+ were competitive inhibitors for variants of MSR. However, 2'5'-ADP was relative strong inhibitor than NADP+. All the data on variants of MSR suggested the Asp652 and tryptophan697 were two important structural and function determinant of MSR. To investigate the dynamic properties of EPR, ENDOR and ESMME are used to investigate the existence of the semiquinone flavin cofactors, FAD and FMN, and the hyperfine coupling arising from the interaction of some nuclei with the unpaired electron spin. ELDOR spectroscopy was applied to measure the distance between the FAD and FMN in MSR under the binding of 2', 5'-ADP, NADP and the activation domain of MS to further check the conformational change of MSR.

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The non-enveloped, pH- and heat resistant rotavirus (RV), which is cross species-infective among cattle, swine and humans may cause dehydration and high mortality in the young. Rotaviruses are inactivated only by corrosive and toxic disinfectants. In this study, the effects of bacterial proteases as a new type of disinfectants on simian rotavirus (SA-11) were analyzed. SA-11 rotavirus replicates in cells causing cytopathic effect (CPE) and is similar in protein composition to cattle and swine RV. Preliminary experiments tested the temperature and pH sensitivity of SA-11 rotavirus. At pH 8.5, 45°C was the highest temperature at which no loss in viral titer was seen, and the virus was still infective following treatment at 65°C for 2 hrs. pH sensitivity tests were then conducted for two hours at 45°C, with pH 5 being the lowest and pH 8.5 being the highest at which no loss in titer was observed. Four proteases were then tested for effectiveness at inactivating SA-11 rotavirus at their pH optimal at 45°C. Alcalase was selected as the most efficient protease. Alcalase was found to inactivate SA-11 at 25°C, and pH 8.5 in 3 days, indicating that enzymes were relatively effective at lower temperatures. SA-11 rotavirus virus was then tested for sensitivity to pH at 25°C and 15°C in absence of enzyme. At pH 2, 25°C a ~4 log reduction was seen following 15 min of treatment, with viable virus still remaining after 8 hrs, at 15°C a ~1.75 log reduction was seen following 2 hrs, and a ~4 log reduction following 8 hrs of treatment. At pH 4 and 6, at 25°C and 15°C no effect on SA-11 titer was seen after 120 hrs treatment. The enzyme was then tested at 1.0% and 0.1% enzyme concentration, at 15°C and 25°C, and pH 6 to determine efficacy of enzyme at sub-optimal conditions. Following treatment with 1.0% Alcalase at 25°C a ~3.25 log reduction, and at 15°C, 1.0% Alcalase, a ~1.75 log reduction was seen at 120 hrs. At 15°C, 1.0% Alcalase a ~1.75 log reduction was seen at 120 hrs. Treatment with 0.1% Alcalase at 25°C, pH 6 resulted in ~2.25 log reduction after 120 hrs. At 15°C, 0.1% Alcalase a ~1.25 log reduction was seen following 120 hrs. The results showed that proteases, used as viral disinfectants, were not as effective at inactivating rotaviruses under simulated field conditions as originally hoped, nevertheless the ease of application and moderate but definite efficacy against rotaviruses may help reduce rotaviral infections and severity of clinical signs in young animals. / Master of Science

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No description available.

No description available.

𝘐𝘯 𝘷𝘪𝘵𝘳𝘰 selection has been widely used to isolate single-stranded DNA molecules from large random-sequence pools that are able to perform a desired catalytic reaction, or bind to a target molecule. Using this technique, we have created a DNA enzyme, named DET22-18, which has a uniquely linked chemical catalysis/real-time signaling capability. It is a true enzyme with a 𝘬cₐₜ of~7 min⁻¹ -the second fastest rate ever reported for a DNA enzyme. The DNA enzyme cleaves a substrate containing a single ribonucleotide linkage embedded in a DNA chain and sandwiched between a fluorophore-labeled deoxyribonucleotide and a quencher-modified deoxyribonucleotide. The ability of DET22-18 to generate a large fluorescence signal provides a useful tool to engineer potential allosteric deoxyribozyme biosensors for real-time detection of important biological targets. To provide a proof of concept that a reporter system can be built from the above signaling DNA enzyme, the cis-acting version of this enzyme, DEC22-18A, was engineered into an allosterically regulated deoxyribozyme biosensor that can report ATP. A preliminary investigation was also conducted to determine a possible secondary structure of the DNA enzyme. This study lays a foundation for pursuing novel signaling DNA enzymes for biological detection directed applications. / Thesis / Master of Science (MSc)

Bacterial resistance to the aminoglycoside antibiotics is a major health concern because of the elimination of a therapeutic option for the treatment of nosocomial infections. Clinical resistance is commonly caused by the acquisition of genes that encode an aminoglycoside modifying enzyme. These enzymes offer a potential therapeutic target in the fight against aminoglycoside resistance. By gaining a structural understanding of these enzymes the potential is created for rational drug design. The research presented here deals with structural studies on two aminoglycoside resistance enzymes. First the initial stages of structural determination for the bifunctional Aminoglycoside 6'-N-Acetyl transferase Aminoglycoside 2''-O-Phosphotransferase (AAC(6')-APH(2")) including the optimization of the purification procedure for this enzyme. The second enzyme is the Aminoglycoside 3'-O-Phosphotransferase (APH(3')IIIa). Computational studies on this enzyme have been carried out in order to determine models for aminoglycoside binding and also to search for potential enzyme inhibitors. The molecular docking studies for both the aminoglycoside binding and inhibitor search involved the development of a number of novel methods to improve the chance of obtaining a correct model, and to aid in the analysis of the data from the docking studies. These methods have the potential to be applied in future structure based drug design / Thesis / Master of Science (MS)