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Subject: search.filters.subject.310106 Enzymes

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    The analysis of inquiry in students' conversations in the biochemistry laboratory : the elucidation of proton-coupled electron-transfer reaction mechanism in manganese superoxide dismutase through structural analysis of mutants : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Hermawan, Jatnika
    Superoxide dismutases (SODs) have very significant biological importance, protecting organisms against reactive oxygen species such as superoxide. They are also known as the fastest enzyme with the largest kcat/Km of any known enzyme. To perform super-fast enzymatic function, SOD must shuttle proton-coupled electrons in an efficient systematic way. However, since its discovery in 1968, the mechanistic nature of SOD catalytic function remains vague. Wide-ranging approaches have attempted to uncover the catalytic mechanism of the manganese-containing SOD, MnSOD, but there were experimental limitations that obstructed the investigations. Here, the structural analyses of two dimer interface mutants of MnSOD, S126D and S126W, explored possible changes in water structure near the active site providing new information to examine the hypothesis of the Glu170 bridge as a key player in the proton shuttle in the outer-sphere mechanism. To gain insight into the mechanism of the proton-coupled electron-transfer (PCET) reaction mechanism, the technique of single-crystal X-ray crystallography was used to observe the three-dimensional structure of Escherichia coli MnSOD mutants, analytical ultracentrifugation was used to observe quaternary association in solution, and protein stability was assessed by differential scanning calorimetry. The key residue Ser126 at the conserved but asymmetric dimer interface of the MnSOD was mutated with the initial intent to generate a monomeric species. Ser126 is not essential for activity and is not part of the active site, whereas Glu170 forms part of the dimer interface where Glu170 from one subunit forms part of the active site of the second subunit of the dimer. The loss of activity occurring in a monomeric MnSOD may indicate an alternative catalytic mechanism of the MnSOD enzyme. The substitution of Ser126 to Asp, intended to produce a monomeric species by charge repulsion, surprisingly produced a dimer at pH>7.5 with little change in structure at the Mn active site, but there was a 94 % reduction in catalytic activity. Partial loss of activity in Ec-MnSOD-S126D may be due to electrostatic effects of the negative charge ~7 Å from metal centre perturbing the Mnᴵᴵᴵ/Mnᴵᴵ redox couple. The substitution of Ser126 to Trp, intended to produce a monomeric species by steric bulk, enforces mostly monomeric Ec-MnSOD S126W in solution form, coupled with a 99.9 % reduction in catalytic activity. Here one mutation to a conserved dimer interface led to altered tertiary structure and a completely different dodecameric domain-swapped quaternary association in the crystalline state and complete loss of activity in Ec-MnSOD-S126W in the solution state. In the course of evolution, higher and less often lower degrees of oligomerisation have arisen. Evolving complexity does not require multiple mutations. As part of the scholarship requirements, this dissertation contains a pedagogical component. Student conversations in a guided inquiry third-year biochemistry laboratory were recorded and analysed to discover the extent of higher-order critical thinking that might occur. Although students initially struggled to move beyond core first-year laboratory skills, they were at all times strongly engaged in the project-style experiment, which ran over three five- to eight-hour sessions. Some progress in the level of inquiry was captured from their conversations from the first to the third laboratory session. A simple diagram and table were developed to help guide teachers in a guided inquiry-based learning in higher education.
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    Identification and characterisation of an enzyme from Monoglobus pectinilyticus associated with degradation of pectin from kiwiberry : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Lewis, Aymee
    Pectin is a complex polysaccharide and a very important source of dietary soluble fibre, present in a variety of fruit and vegetables. Pectin possesses a wide range of contributions to a healthy diet and a healthy human gut, such as lowering cholesterol, protecting against intestinal inflammation and maintaining digestive health. Yet pectin is unable to be degraded by human gastrointestinal enzymes, arriving in the large intestine mostly intact. Limited research has showed the domination of Gram-negative bacteria which possess a range of secreted extracellular cell-bound and cell-free pectin degrading enzymes. These enzymes attack the pectin backbone and the accessory side chains resulting in the isolation of mono/oligosaccharides for subsequent uptake. The recent novel discovery of Monoglobus pectinilyticus, a Gram-positive bacterium, expanded the narrow knowledge regarding specific pectinolytic degraders. Genomic studies showed this bacterium contains an arsenal of enzymes specifically for pectin degradation. However, several putative pectin-degrading enzymes produced by M. pectinilyticus were unable to be identified against up-to-date databases, thus suggesting that there may be potentially novel classes of CAZyme(s) to be discovered. The proposed study will attempt to identify such enzyme(s) using New Zealand cultivar kiwiberries as the source of pectin. The initial aim of this study was to investigate the enzyme(s) involved in β-1,4-galactan by growing M. pectinilyticus with a mixture of pectin substrates (0.5% citrus pectin, 0.5% kiwiberry pectin and 0.5% potato galactan), in hope to identify such enzyme(s). Unfortunately, no β-galactosidase/β-galactanase was found. However a potentially novel binding enzyme, protein 0050 was isolated, cloned and expressed, which may be involved in a pectin degrading version of a cellulosome complex. Further research into isolating a variety of secreted proteins activated in presence of galactan during M. pectinilyticus cultivation is needed to understand the complexity of pectin degradation.
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    Investigating the role of HDAC4 in Drosophila neuronal function : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand
    (Massey University, 2022) Tan, Wei Jun
    HDAC4 plays an essential role in brain functions including neurodevelopment and memory formation, and increased levels of HDAC4 have also been associated with neurodegenerative disorders including Alzheimer’s disease. Histone deacetylases are enzymes that are traditionally known to regulate gene expression in the nucleus, however in neurons, HDAC4 shuttles between the nucleus and cytoplasm with a predominant distribution in the cytoplasm. Although studies have identified potential differences in subcellular function in which accumulation of nuclear HDAC4 has been shown to promote neurodegeneration, while cytoplasmic HDAC4 is neuroprotective, the mechanistic pathways through which it acts are still unknown. Therefore, this project aimed to determine the importance of nuclear and cytoplasmic pools of HDAC4 to the neurological functions of Drosophila melanogaster, as well as to determine the domains within the protein that are required for its function(s). This was carried out by expressing HDAC4 with mutations that resulted in altered subcellular distribution or carrying mutations in binding domain/motifs that have previously been shown to be important for HDAC4 function. Increased expression of wild-type HDAC4 disrupted development of the retina and the mushroom body (MB, a brain structure derived from Kenyon cells which are crucial for learning and memory), and expression of each mutant revealed the importance of specific domains/motifs to HDAC4 function in these tissues. Of interest, impairments to MB formation were exacerbated by mutation of the ankyrin-binding site and by mutation of serine residues that promote nuclear exit when phosphorylated (i.e. resulting in restriction to the nucleus). Mutation of the MEF2-binding site ameliorated these phenotypes, suggesting that HDAC4 acts through MEF2 to regulate MB development. However, while deacetylase activity was found to be dispensable in the MB, an active deacetylase domain was required in order for the phenotype to manifest in the retina, and mutation of the MEF2-binding site had no impact on the deficits caused by nuclear restriction of HDAC4 and mutation of the ankyrin-binding domain. Together these data indicate that HDAC4 acts through varying mechanism(s) depending on the cell type. Transcriptional changes in the Drosophila brain resulting from the expression of HDAC4 or its mutant variants was also explored using RNA-Seq. However only wild-type HDAC4 resulted in a large number of differentially expressed genes and the low level of differential gene expression in HDAC4 variants suggests that non-transcriptional processes may be involved in the induction of phenotypes caused by expression of these mutants. Additionally, further analysis of genes that were differentially regulated revealed a number of processes related to mitochondrial energy production. These findings have provided new insights into the role of HDAC4 in Drosophila neurodevelopment which opens up additional research avenues to focus on in the future.
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    Investigating the role of herzog in the Drosophila melanogaster brain : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2021) Palmer, Madeleine
    Memory and cognitive disorders, such as Alzheimer's disease and age-associated memory loss, are common, but currently there is no cure or effective treatment. Better understanding of the pathways involved in memory and how disruptions within these occur may assist in the development of new treatments. To further dissect normal memory and how disruptions in these pathways affect long-term memory, it is vital to first understand which proteins are involved and their function within memory. Histone deacetylase 4 (HDAC4) plays an important role in memory and brain development in Drosophila melanogaster. Mutations in HDAC4 result in intellectual disability in humans and overexpression causes memory deficit in Drosophila. The phosphatase herzog was identified as a potential gene target of HDAC4 via a previous RNA-seq experiment. We found herzog to be highly expressed in the brain, especially in the mushroom body. No nuclear localisation of herzog was seen which contrasts to the localisation of human CTDSP1 suggesting they have diverged in regard to their functions and phosphatase targets. Drosophila eye development requires herzog, however mushroom body development, courtship learning and short-term memory does not require herzog.
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    Biophysical and biochemical characterisation of DNA-based inhibitors of the cytosine-mutating APOBEC3 enzymes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2020) Barzak, Fareeda Maged Yahya Mohammad
    With the rise of antiviral and anticancer drug resistance, a new approach must be taken to overcome this burden. The APOBEC3 (A3) family of cytosine deaminases hypermutate cytosines to uracils in single-stranded DNA (ssDNA). These enzymes act as double-edged swords: on one side they protect humans against a range of retroviruses and other pathogens, but several A3s are exploited by viruses and cancer cells to increase their rate of evolution using the enzyme’s mutagenic actions. This latter mode permits escape of cancer cells from the adaptive immune response and leads to the development of drug resistance. In particular, APOBEC3B (A3B) is considered to be a main driving source of genomic mutations in cancer cells. Inhibition of A3B, while retaining the beneficial actions of the other A3 in the immune system, may be used to augment existing anticancer therapies. In this study, we showed for the first time that short ssDNAs containing cytosine analogue nucleosides, 2'-deoxyzebularine (dZ) or 5-fluoro-2'-deoxyzebularine (5FdZ) in place of the substrate 2'-deoxycytidine (dC) in the preferred 5'-TC motif, inhibit the catalytic activity of A3B. However, as most A3 enzymes (except A3G) prefer to deaminate ssDNA with a 5'-TC motif, selective A3B inhibition was uncertain. We noted that nucleotides adjacent to the 5'-CCC motif influence the dC deamination preference of A3A, A3B, and A3G’s. Replacement of the A3B’s preferred dC in the 5'-CCC motif with dZ (5'-dZCC) led to the first selective inhibitor of A3B, in preference to A3A and A3G. Furthermore, using small-angle X-ray scattering (SAXS) we obtained the first model of a full-length two-domain A3 in complex with a dZ-ssDNA inhibitor. Our model showed that the ssDNA was largely bound to the C-terminal domain (CTD) with limited contact to the N-terminal domain in solution, due to the high affinity of the dZ for the CTD active-site. Our work provides a new platform for use of ssDNA-based inhibitors in targeting the mutagenic action of the A3B. Further developments using more potent inhibitors will help to achieve inhibition in cellular studies, with the ultimate goal to complement anti-cancer and antiviral treatments.
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    Studies toward evaluating Gcn2 as a drug target : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany, New Zealand. EMBARGOED until 24 March 2027.
    (Massey University, 2020) Prescott, Hayley
    All living organisms are subject to dynamic environmental conditions. In order to survive a changing environment, organisms have evolved the capacity to adapt to stressors; this adaptation response occurs on both physiological and cellular levels. The majority of organisms face periods of inconsistent nutrient supply, which require cells to regulate consumption and conservation of vital resources such as amino acids and glucose. Amino acids are a fundamental nutrient essential for macromolecule biosynthesis; they are the monomeric unit of proteins, required for practically every cellular function. The amino acid stress response pathway regulates amino acid conservation when intracellular amino acid depletion occurs under physiological and pathological conditions; a key constituent of this pathway is Gcn2. Gcn2 is a ubiquitous eukaryotic protein kinase. Gcn2 is necessary for the maintenance of cellular homeostasis in response to amino acid deprivation and other physiological stressors such as UV irradiation and glucose, purine, or carbohydrate deprivation; Gcn2 not only aids survival in response to these regular physiological events but, on the other hand, is crucial for the progression of some pathologies, which include cancer. Gcn2 activity contributes to the growth and maintenance of tumours; it is, therefore, recognised as a prospective anti-cancer drug target. Inhibition of Gcn2 may be an effective strategy for impeding cancer cell proliferation and tumour growth as a stand-alone therapy or as a component of a combined therapeutic approach. In this study, we combined computational and experimental analytical methods to identify Gcn2 inhibitors and examined the effects of Gcn2 inhibition on cancer cells in combination with currently used cancer therapies.--Shortened abstract
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    Studying the relationship between emulsion structure and lipid digestibility for infant milk : a thesis was present in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology, at Massey University, Palmerston North, New Zealand
    (Massey University, 2020) Deng, Le
    Milk, whether maternal or formulated, provides the sole source of nutrition to infants in the early stages of life, providing critical micronutrients, support for the immune function and primary dietary macronutrients including lipids. In healthy adults, lipids are primarily digested in the small intestine. However, for infants, the neonatal small intestine is not fully developed after birth, so the gastric environment plays a more significant role in milk fat digestion. Clinical studies have shown that maternal milk fat is digested more efficiently than lipids in infant formulae in infants under infant gastric conditions. Compositional differences, the structure of the oil droplets, and especially the interfacial composition may all play a crucial role in influencing lipid digestibility in the infant's stomach. In this thesis, the simulated gastric digestion of model emulsions and commercial infant formula was studied. The model emulsions comprised either a phospholipid or complexed protein-phospholipid interface while keeping all other facets of emulsion properties equivalent. Gastric digestion of these emulsions was carried out across variable pH conditions using an analogue gastric lipase, alone and in combination with pepsin with findings providing insights into the role of each enzyme and their combined effect on gastric lipolysis. The rate and extent of lipolysis were characterised, along with morphological changes to the structure of the oil droplets. Results showed that gastric lipolysis might be influenced by pH conditions in the gastric environment when lipase was present alone in the simulated gastric fluid. The inclusion of pepsin resulted in significant structural changes when emulsions were stabilised with protein, in terms of droplet aggregation, size and morphology. However, no significant differences in the extent of lipolysis were determined. Thus, while the protein interface of both model and formulated emulsions was not observed to be a barrier for gastric lipolysis. Proteolysis of protein stabilised emulsions may lead to very different structural outcomes during gastric digestion when compared to phospholipid stabilised emulsions. While the research within this thesis demonstrates how the gastric environment influences emulsion structure as a consequence of interfacial composition, any specific relationship between structure and relative rate of gastric lipolysis currently remains undetermined. This research also highlights some of the ongoing challenges in the use of in vitro models to provide mechanistic understanding and interpretation of findings from clinical studies.