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Characterization of multicopper oxidase-related protein and multicopper oxidase-1 in insectsPeng, Zeyu January 1900 (has links)
Doctor of Philosophy / Biochemistry and Molecular Biophysics / Michael R. Kanost / Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms, which are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence identity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We purified recombinant Tribolium castaneum (red flour beetle) MCORP. As expected, no oxidase activity was detected. We analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP. They are constitutively expressed at a low level in many tissues, including ovaries. TcMCORP larval RNAi led to 100% mortality before adult stage. These deaths occurred during the larval to pupal and pupal to adult molts. Pharate pupal RNAi resulted in 20% mortality during the pupal to adult molt, and 100% mortality by one month after adult eclosion. In addition, knockdown of TcMCORP in females prevented oocyte maturation, thus greatly decreasing the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests.
A multicopper oxidase-1 (MCO1) ortholog has been identified in all insect species examined so far; thus, MCO1 probably has a conserved physiological function in insects. Most of the well-studied MCOs are laccases, ferroxidases, or ascorbate oxidases. Previously we found Drosophila melanogaster MCO1 has ferroxidase activity and we identified three putative iron binding residues in DmMCO1. Our kinetic analysis of recombinant MCO1 from Drosophila melanogaster, Anopheles gambiae, Tribolium castaneum and Manduca sexta showed that MCO1 orthologs are much better at oxidizing ascorbate than laccase substrates or ferrous iron, suggesting that MCO1 orthologs function as ascorbate oxidases. The putative iron binding residues are required for ascorbate oxidase activity but not ferroxidase and laccase activities. Ascorbate oxidases have been identified only in plants. This is the first identification of ascorbate oxidase in insects. Further studies are needed to understand their physiological function in insects.
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Mechanisms of Bacterial Copper Detoxification and Oxygen Reduction in CueO and Chemotactic Signal Amplification by Receptor ClusteringSingh, Satish Kumar January 2009 (has links)
CueO is a multicopper oxidase and catalyses the four-electron reduction of dioxygen to water and functions to protect Escherichia coli against copper-induced toxicity. The mechanism of oxygen reduction in multicopper oxidases has been well studied, but the key structures of the reaction intermediates are not known. A combination of kinetic measurements, mutagenesis and X-ray crystallographic studies were conducted to entrap and structurally characterize the reaction states in CueO. CueO has a methionine-rich insert and a labile copper binding site, two features found only in multicopper oxidases involved in copper detoxification. The role of these features in CueO activity has been investigated. In a separate study, a simple mathematical model based on infectivity amongst clustered receptors was developed to explain the chemotactic sensitivity, response range and other key features of chemotaxis.This study describes the successful entrapment of three out of four functional states in CueO. The crystal structures of these reaction states are presented. Using single-turnover oxygen reduction kinetics that were measured using a stopped-flow device, the optical absorption features of three different fully oxidized forms of CueO were captured: the native intermediate, the resting oxidized state and another intermediate lying between them. Stopped-flow studies combined with electron transfer kinetic measurements revealed a role of the conserved residue, E506, in either the protonation of the native intermediate or the release of water molecules formed as a product of the reaction.Cu(I) and Ag(I) bound crystal structures of CueO were determined revealing three binding sites along the methionine-rich helix used by both metal ions. The labile, regulatory copper site in CueO was shown to be a Cu(I) susbtrate oxidation site. Ag(I) was shown to be a potent inhibitor of all CueO activities in vitro and copper detoxification by the cue system in vivo. The cus system was discovered to be necessary for removing Ag(I) inhibition of copper detoxification by the cue system. These results provide further insights into the role of CueO in copper detoxification and the effect of silver on the detoxification mechanism.
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Recombinant expression and characterization of two isoforms of Anopheles gambiae laccase-2Sullivan, Lucinda I. January 1900 (has links)
Master of Science / Department of Biochemistry / Michael R. Kanost / Laccases are multicopper oxidases that catalyze the oxidation of a broad range of substrates, typically phenols and anilines. Research on laccases in fungi, plants, and bacteria has indicated that they have roles in detoxification, pigmentation, wound healing, morphogenesis and lignin synthesis and degradation. However, there has been relatively little investigation on laccases that exist in insects or other invertebrates. Insects have multiple laccase genes, but the function of just one type is known; laccase-2 (Lac2) orthologs are required for tanning of newly synthesized exoskeleton. In the mosquito Anopheles gambiae and other insect species whose genomes have been sequenced, alternative exon splicing may generate two isoforms of Lac2. The objective of this study was to characterize the two isoforms of AgLac2. They are identical in their first 500 residues, but the carboxyl-terminal 262 residues derived from alternative exons are 81% identical. Recombinant Lac2A and Lac2B were expressed and purified. They are both glycoproteins of ~81 kDa, and both can oxidize the laccase substrate ABTS as well as the catechols, N-β-alanyldopamine (NBAD) and N-acetyldopamine (NADA). Lac2A and Lac2B with ABTS have pH optima of 5.0-5.5 and 4.5-5.0, respectively. The pH optima with NBAD and NADA are 5.5-6.5. The Km values (mM) for Lac2A and Lac2B with NBAD are 5.4 ± 2.1 and 5.0 ± 2.6, respectively. The Km values (mM) for Lac2A and Lac2B with NADA are 0.7 ± 0.2 and 1.4 ± 0.5, respectively. Thus, there is little difference between the isoforms in K[subscript]m for these two substrates. The K[subscript]m values do indicate that both isoforms have a greater affinity for the substrate NADA. The kcat values (s[superscript]-1) for Lac2A and Lac2B with NBAD are 14.2 ± 3.5 and 6.0 ± 1.8, respectively. The k[subscript]cat values (s[superscript]-1) for Lac2A and Lac2B with NADA are 2.4 ± 0.2 and 0.5 ± 0.04, respectively. The most apparent difference between the two isoforms detected in the study is that Lac2A was four-fold more active than Lac2B when NADA was used as a substrate. Although the two isoforms are very similar in their amino acid sequences, the differences in catalytic properties may indicate different roles in insect physiology.
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Application of synthetic tricopper complexes and NOx in energy conversion and storageZhang, Weiyao 04 November 2022 (has links)
No description available.
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