Although transition metals are essential for life, misregulation of redox-active metal uptake, delivery, storage, and excretion has been linked with a series of neurodegenerative disorders. Alzheimer's disease (AD) is considered an epidemic and is the most widespread of all forms of dementia. Copper ions found in large concentrations localized in amyloid-ß plaques in the brain of AD patients have been linked with the generation of reactive oxygen species which are suspected to be the culprits leading to neuronal cell death. Herein a series of mechanistic and spectroscopic studies elucidate the chemistry about the metal-centered oxidation of biomolecules, including catecholamine neurotransmitters and some analogues by copper-complexes of amyloid-ß peptide. Transition metals can also be useful tools for characterization of metalloproteins due to their unique chemical and spectroscopic features. Herein a series of studies of the native Zn²+ and Cu²+-derivative of recombinant Blastula Protease 10 (BP10) from the sea urchin Paracentrotus lividus are presented in order to elucidate its catalytic mechanism, with the use of enzymology, metal substitution, and electronic absorption spectroscopy.

Functional metagenomic approach was performed using total environmental DNA extracted from a temperate forest soil sample and from an Antarctica soil sample. Searching for clones harbouring phenotypes related to the production of new hydrolytic enzyme allows the isolation of several new enzymes. Amongst them, an esterase and a cellulase, named RBest1 and RBcel1 respectively, were characterized. By accurate description of their catalytic proprieties these two new enzymes appear to present interesting features. The RBest1 esterase is an enzyme whose activity is stabilised or improved in presence of non water-miscible organic solvent. By sequence analysis, RBest1 is related to other organic solvent tolerant enzyme. Moreover, in aqueous buffer, RBest1 is highly specific for butyrate compound but surprisingly its specificity appears to be shifted in presence of organic solvent. The RBcel1 cellulase, was thoroughly characterized for its involvement both in cellulose degradation and production. Our data highlight the requirement for such enzyme in the bacterial cellulose synthesis process. According to our results, the mining of metagenomic libraries by functional screening associated to detailed description of the isolated enzymes gives hints for both ecological and microbiological questions.

<p>Sulfite oxidase, a metabolically important enzyme, catalyzes the physiologically critical conversion of sulfite to sulfate in the terminal step of the degradation of sulfur containing compounds. The enzyme has been the focus for much research since its discovery in the 1950's. A central question to understanding the mechanism of molybdoenzymes such as sulfite oxidase and nitrate reductase concerns the roles of active site residues and the coordination chemistry of the Mo atom in the structure and function of the enzyme. The goal of this work was directed towards the characterization and determination of the structures of active site variants of sulfite oxidase using a spectroscopic, kinetic, and protein crystallographic approach.</p><p>Earlier studies have identified a single, highly conserved cysteine residue as the donor of a covalent bond from the protein to molybdenum in sulfite oxidase and nitrate reductase. The C185S and C185A variants of chicken sulfite oxidase exhibited severely attenuated activity. Crystallographic and spectroscopic analysis of both variants revealed a change in the metal coordination, from a dioxo to a trioxo form of Mo. </p><p>Assimilatory nitrate reductase is a member of the sulfite oxidase family of molybdopterin enzymes. The crystal structure of the Mo domain of the enzyme from Pichia angusta revealed high structural homology in the active sites of nitrate reductase and sulfite oxidase. Both enzymes utilize the same form of the molybdenum cofactor and have three out of five residues conserved at the active site. Substitution of two active site residues in sulfite oxidase alters the substrate affinity of chicken SO from sulfite to nitrate, resulting in an increase of nitrate reductase activity over wild-type sulfite oxidase. Additionally we identified an additional amino acid position in sulfite oxidase that corresponds to a non-conserved position in NR that further increased NR activity. Finally, these nitrate reductase variants of sulfite oxidase were crystallized and the structures solved. This represents the first example of the transmutation of a molybdenum enzyme to change activity and substrate affinity to those of a homologous enzyme.</p> / Dissertation

The primary goals of our research are to understand the virology and enzymology of human T-cell leukemia virus type I (HTLV-I) that will lead to the development of treatments for patients infected with HTLV-I. HTLV-I is an oncogenic virus of the Retroviridae family and is the causative agent of adult T-cell leukemia/lymphoma (ATL), tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). HTLV-I has been classified as a dangerous emerging pathogen by the Centers for Disease Control and Prevention with at least 20 million people infected with the virus. This is a significant problem because there are currently no effective treatments to control HTLV-I infection and prevent or treat HTLV-I induced ATL and TSP/HAM. The protease is necessary for retroviral maturation and replication and is, therefore, an attractive target for inhibitor design. Investigation of peptide mimetic compounds incorporating the tetrahedral intermediate of aspartyl protease catalyzed cleavage are crucial for the development of lead inhibitors. Compounds containing statine, 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), or hydroxyethylamine (HEA) are presented in this work. The best compound was a statine-based inhibitor, which had a Ki = 29 +/- 4 nM and 88% inhibition against an HTLV-I protease native substrate in a FRET assay.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 180-198). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 132-138).

Nitric oxide synthases (NOS) are responsible for the production of nitric oxide (NO), an essential cell-signaling molecule, in mammals. There are three isoforms of NOS with widely different tissue distribution. The overproduction of NO is marked in many human disease states and cancers, however due to the similarities of the enzyme isoforms, targeting NOS for inhibition has proven challenging. Endogenously, the methylated arginines, N[superscript ω]-monomethyl-L-arginine (NMMA) and asymmetric N[superscript ω], N[superscript ω]-dimethyl-L-arginine (ADMA), inhibit NOS. N[superscript ω], N[superscript ω]-Dimethylarginine dimethylaminohydrolase (DDAH1) metabolizes these methylated arginines and thus relieves NOS inhibition. The role of DDAH1 in the regulation of diseases such as cancer and septic shock is still being elucidated. It is thought that targeting DDAH1 for inhibition rather than NOS may circumvent many of the current problems with the treatment of NO overproduction such as isoform selectivity. My PhD studies focus on the synthesis of a series of irreversible inhibitors of DDAH1, an extensive study of their in vitro mode of inhibition, a comparison of analytical fitting methods, and the viability and efficacy of the inactivators in a human cell line. I also studied a potential endogenous inactivator of DDAH1, nitroxyl (HNO), a one-electron reduction product of NO. / text

Type II polyketide synthases (PKSs) are responsible for the generation of structurally diverse and clinically important aromatic polyketides. The bacteriostatic agent enterocin (enc) isolated from the marine microbe "Streptomyces maritimus" is derived from a rare benzoate primer unit and contains a unique nonaromatic caged core structure resulting from a Favorskii-like carbon skeleton rearrangement. The apparent diversion between enterocin biosynthesis and all other type II PKS pathways offered the opportunity to discover novel enzymatic strategies that may be exploited to diversify the chemical structures of polyketides. A comprehensive biochemical analysis was performed in order to characterize the key steps in enterocin biosynthesis and finally to reconstitute the whole pathway in vitro using purified recombinant enzymes.A nonribosomal peptide synthetase (NRPS)-like priming mechanism was discovered for the selective activation of a benzoic acid starter unit and its subsequent attachment to the enc PKS to initiate polyketide biosynthesis. This is the first example of a type II PKS that employs an NPRS-like priming mechanism to utilize alternative non- acetate starter units. Secondly, the minimal enc PKS was reconstituted in vitro to give three novel acetate-primed metabolites that had never been identified by heterologous in vivo expression of recombinant enc PKS gene sets. The minimal enc PKS was then merged with the NRPS-like chain initiation module and the resulting multienzyme complex catalyzed the formation of benzoate-primed natural products wailupemycin F and wailupemycin G. Favorskii-like rearrangement of the nascent polyketide chain was replicated in vitro and the flavin-dependent oxygenase EncM was confirmed to be solely responsible for catalyzing this unprecedented rearrangement. Other biosynthetic steps in the late stage of the enc pathway were also replicated in vitro, including the methylation of desmethyl-5-deoxyenterocin to 5-deoxyenterocin and the hydroxylation of 5-deoxyenterocin to enterocin.Finally, the entire enc type II PKS pathway was successfully assembled in vitro using ten recombinant proteins and three commercial enzymes. Five enc-based natural products were generated from benzoic acid and malonyl-coenzyme A. This biochemical investigation on enterocin biosynthesis represents the first complete in vitro reconstitution of a type II PKS system and also provides an alternative strategy to create complex natural products by multienzyme synthesis.

Septic shock is a leading cause of death in intensive care units. As part of the septic process, the endothelium becomes activated and propagates the septic condition. It has become evident that reactive oxygen species (ROS) are involved in the signaling of mediators of sepsis, such as tumor necrosis factor-alpha (TNF-alpha) and the lipopolysaccharide coating of gram-negative bacteria (LPS). An important source of these ROS is NADPH oxidase, which is a ubiquitously expressed enzyme complex that also exists in endothelial cells (EC). We showed that O2- from NADPH oxidase was important for LPS, as well as TNF-alpha, induction of two markers of an activated endothelium, interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVEC). / Expression of a gene can be increased by a rise in transcription as well as post-transcriptional changes, such as mRNA stability modifications. We assessed the role of NADPH oxidase in this process and found a complex interaction. Although LPS increases IL-8 transcription, it also destabilizes IL-8 mRNA in a p38 and extracellular signal-regulated kinase (ERK) MAPK dependent manner, which was only evident after blocking NADPH oxidase. This regulation involved the mRNA de-stabilizing factor tristetraprolin (TTP). In contrast, TNF-alpha enhanced the stability of IL-8, IL-6 and ICAM-1 mRNA in a p38 MAPK dependent, but NADPH oxidase independent manner. Furthermore, LPS did not have an effect on mRNA stability of IL-6 or ICAM-1 in our system. Thus, we conclude from our studies that the NAPDH oxidase is important for the induction of inflammatory molecules in LPS and TNF-alpha treated EC and is also involved in mRNA stability regulation of these molecules in a signal and gene specific fashion.

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury in both native kidneys and renal allografts. Previous studies in our lab have shown that a subset of Eph family receptor tyrosine kinases, including EphA2, is strongly and persistently upregulated in renal tubular cells in both in vitro and in vivo models of the renal IRI. Src kinases are necessary and sufficient for upregulation of EphA2. We have proposed that IRI-induced EphA2 upregulation may serve as a necessary step in renal tubular remodelling. / In this study, we have further defined the mechanism of Src kinase-induced EphA2 upregulation by identifying the -145/+137 EphA2 promoter region as the minimal region required for basal and Src kinase-induced activation of the promoter. Moreover, we have identified within this region, at position -45, a canonical cAMP response element (CRE) (Nowakowski et al.), which is essential for EphA2 promoter activation. However, we also found that the prototypical CRE-binding transcription factor, CREB, was not necessary for activation of the EphA2 promoter, suggesting that CREB-related or -unrelated transcription factors are responsible for EphA2 upregulation.