Life Sciences Commons^™

The Discovery And Analysis Of Peroxidase Enzyme In Pueraria Montana, Kathryn Briley

Senior Honors Theses

Peroxidase enzymes are used for a variety of industrial and biotechnological applications because of their ease of purification, broad range of chemical activities, and low cost of use. Identification of quality peroxidase sources that are convenient for enzymatic isolation and give way to high yields of product is a desirable pursuit in biochemical research. Kudzu is an excellent candidate for this pursuit as it displays high catalytic activity in screening assays and is found in abundance. This project seeks to determine the enzyme stability, optimal pH conditions, and possible novel chemical activities of peroxidase isolated from kudzu leaves. The methods …

Transition State Interactions In A Promiscuous Enzyme: Sulfate And Phosphate Monoester Hydrolysis By Pseudomonas Aeruginosa Arylsulfatase, Bert Van Loo, Ryan Berry, Usa Boonyuen, Mark F. Mohamed, Marko Golicnik, Alvan C. Hengge, Florian Hollfelder

Chemistry and Biochemistry Faculty Publications

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PAS^_WT has a much less negative Brønsted coefficient (ß_{leaving group} ^obs-Enz=-0.33) than the uncatalyzed reaction (ß_{leaving group} ^obs=-1.81). This situation is diminished when cationic active site groups are exchanged for alanine. …

Mutant Study Of Sinorhizobium Meliloti Proline Utilization A (Puta), Jacob E. Wilkinson, John J. Tanner, Donald F. Becker

UCARE Research Products

The purpose of this project is to purify and characterize the reaction kinetics of mutant versions the enzyme Proline Utilization A (PutA) in Sinorhizobium meliloti. The enzyme catalyzes the first step in proline metabolism. It has two active sites. The first is proline dehydrogenase (PRODH) which converts proline to pyrroline-5-carboxylate (P5C). The second is P5C dehydrogenase (P5CDH) which converts P5C to glutamate. Although many bacterial organisms have PutA, there are still significant interspecies variations, resulting in an entire family of PutA enzymes. The main difference is the length of the amino acid sequence. This affects the protein’s structure or …

Amino Acid Racemase Enzyme Assays, Atanas D. Radkov, Luke A. Moe

Plant and Soil Sciences Faculty Publications

Amino acid racemases are enzymes that invert the α-carbon stereochemistry of amino acids (AAs), interconverting amino acids between their L- and D-enantiomers in a reversible reaction. In bacteria, they are known to have catabolic physiological functions but are also involved in the synthesis of many D-AAs, including D-glutamate and D-alanine, which are necessary components of the peptidoglycan layer of the bacterial cell wall. As such, amino acid racemases represent significant targets for the development of bactericidal compounds. Amino acid racemases are also regarded by the biotechnological industry as important catalysts for the production of economically relevant D-AAs. Here, we provide …

Rescuing Acetylcholinesterase From Nerve Agent Inhibition: Protein Dynamics Driven Drug Discovery, Aiyana M. Emigh, Brian Bennion

STAR Program Research Presentations

Severe morbidity and mortality consequences result from irreversible inhibition of human acetylcholinesterase by organophosphates (OPs). Oxime-based reactivators are currently the only available treatments but lack efficacy in the central nervous system (CNS) where the most damage occurs. Computational docking and molecular dynamics (MD) simulations reveal complex structural barriers that may reduce oxime efficacy. These results may guide future drug designs of more effective countermeasures.

Inhibitory Effects Of Novel Immucillin Analogues On Borrelia Burgdorferi Bgp Nucleosidase, Christian Guerrero, Seth Eidemiller, Ken Cornell

IDeA Network for Biomedical Research Excellence (INBRE)

The pathogenic spirochaete Borrelia burgdorferi causes Lyme disease and is transmitted by deer ticks when they feed. Lyme disease is multisystemic—it adversely affects the heart, joints, and skin. Recent studies demonstrate that B. burgdorferi possesses three methylthioadenosine/Sadenosylhomocysteine (MTA/SAH) nucleosidases essential for the catabolic breakdown of both MTA and SAH. Both MTA and SAH are by-products of major pathways involving Sadenosylmethionine (SAM) and are kept at low micromolar concentrations due to their inhibitory activity.

This project examined the effect of transition state inhibitors on the surface binding Borrelia glycosaminoglycanbinding protein (Bgp) nucleosidase using recombinant Bgp and whole-cell B. burgdorferi activity assays. …

Developing A Protocol For Purifying The Mala Enzyme In Bdellovibrio Bacteriovorus, John Jared Trecker

Summer Research

The sequenced genome of the gram-negative predatory bacterium Bdellovibrio bacteriovorus contains a gene that encodes for malA, a putative maltase. Given the bacterium's observed disuse of prey carbohydrates, the gene's presence is mysterious. That characterization of the enzyme and studies of its activity and specificity can be better carried out, it is necessary to obtain pure enzyme. Protein was collected from lysed cultures of Top10/pmalA E. coli. Attempted purification by ion-exchange chromatography with DEAE columns produced significantly purer protein; SP ion exchange columns were unsuccessful, as were heparin and hydroxyapatite affinity columns. Gel filtration chromatography should prove a useful method …

The Primary Structure Of A Fungal Chitin Deacetylase Reveals The Function For Two Bacterial Gene Products., Dimitris Kafetzopoulos, George Thireos, John N. Vournakis, Vassilis Bouriotis

Dartmouth Scholarship

Chitin deacetylase (EC 3.5.1.41) hydrolyzes the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin. A cDNA to the Mucor rouxii mRNA encoding chitin deacetylase was isolated, characterized, and sequenced. Protein sequence comparisons revealed significant similarities of the fungal chitin deacetylase to rhizobial nodB proteins and to an uncharacterized protein encoded by a Bacillus stearothermophilus open reading frame. These data suggest the functional homology of these evolutionarily distant proteins. NodB is a chitooligosaccharide deacetylase essential for the biosynthesis of the bacterial nodulation signals, termed Nod factors. The observed similarity of chitin deacetylase to the B. stearothermophilus gene product suggests that this gene …