Snapshots of an enzyme in action: structural and mechanistic studies on the catalytic cycle of Escherichia coli ketol-acid reductoisomerase. Enzymes are required for almost every process that occurs in a living organism. For this reason, understanding how enzymes work is essential if we are to understand life itself. In this project we will investigate the enzyme KARI by capturing a series of snapshots of its atomic structure as it progresses through its working cycle. In addition, we will make ....Snapshots of an enzyme in action: structural and mechanistic studies on the catalytic cycle of Escherichia coli ketol-acid reductoisomerase. Enzymes are required for almost every process that occurs in a living organism. For this reason, understanding how enzymes work is essential if we are to understand life itself. In this project we will investigate the enzyme KARI by capturing a series of snapshots of its atomic structure as it progresses through its working cycle. In addition, we will make a series of small alterations to the atomic structure that will allow us to understand how the individual parts work together.Read moreRead less
Structure and inhibition of acetohydroxyacid synthase. Acetohydroxyacid synthase (AHAS) has been identified as the target for several widely used herbicides known as the sulfonylureas and imidazolinones. World-wide, these two herbicides account for $US2 billion in annual sales. The aim is to determine the three-dimensional structure of AHAS from several sources and in complex with these herbicides. Furthermore, AHAS appears to be an excellent target for the development of antibacterial compounds ....Structure and inhibition of acetohydroxyacid synthase. Acetohydroxyacid synthase (AHAS) has been identified as the target for several widely used herbicides known as the sulfonylureas and imidazolinones. World-wide, these two herbicides account for $US2 billion in annual sales. The aim is to determine the three-dimensional structure of AHAS from several sources and in complex with these herbicides. Furthermore, AHAS appears to be an excellent target for the development of antibacterial compounds and fungicides. Knowledge of the three dimensional structures of these enzymes will be important in the rational design of more effective inhibitors with improved selectivity.Read moreRead less
Molecular mechanisms of pilin glycosylation in Neisseria: a model system for protein glycosylation in bacteria. The disease causing bacteria Neisseria meningitidis and Neisseria gonorrhoeae are important human pathogens. Cell surface structures, called pili, are known to be important in allowing the bacteria to stick to host cells. Genetic and structural studies have identified that the protein subunits, which make up pili, are glycosylated - modified by the addition of sugars. Until recently ....Molecular mechanisms of pilin glycosylation in Neisseria: a model system for protein glycosylation in bacteria. The disease causing bacteria Neisseria meningitidis and Neisseria gonorrhoeae are important human pathogens. Cell surface structures, called pili, are known to be important in allowing the bacteria to stick to host cells. Genetic and structural studies have identified that the protein subunits, which make up pili, are glycosylated - modified by the addition of sugars. Until recently glycosylation of Gram-negative bacterial proteins was not thought to occur, however our recent work with these bacteria, and other groups studying Pseudomonas and Campylobacter, have shown that this process may be widespread. In our previous studies, we have identified and analysed a number of genes involved in pili glycosylation, in bacteria, which make known sugar structures. We have used this information to developed models for how the biochemistry and physiology of the glycosylation system may work. With a well-established structure and many genes already identified, glycosylation in Neisseria represents the best available model system to study this novel and important process. In the proposed study we describe experiments planned to test our models and reveal the molecular detail of this process. This study could lead to major advances in our understanding of this process and, when understood, may have future applications in biotechnology.Read moreRead less
Mechanistic Studies of Dimethylsulfide Dehydrogenase: A Novel Bacterial Molybdoenzyme. The aim of this proposal is to use electrochemical, spectroscopic and molecular biological techniques to understand the mechanism of action of the enzyme dimethylsulfide dehydrogenase. This enzyme is representative of an major group of molybdenum-containing enzymes that have importance in microbial biotransformations. The project will provide fundamental information about a multi-redox centre protein that has ....Mechanistic Studies of Dimethylsulfide Dehydrogenase: A Novel Bacterial Molybdoenzyme. The aim of this proposal is to use electrochemical, spectroscopic and molecular biological techniques to understand the mechanism of action of the enzyme dimethylsulfide dehydrogenase. This enzyme is representative of an major group of molybdenum-containing enzymes that have importance in microbial biotransformations. The project will provide fundamental information about a multi-redox centre protein that has potential application in biosensors and biocatalysis.Read moreRead less
Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0344441
Funder
Australian Research Council
Funding Amount
$390,000.00
Summary
New Generation Metalloenzyme Magnetic Circular Dichroism Spectrometer Systems. Funding is sought to enhance the existing collaborations between UQ, ANU, Sydney and other universities in the study of metal-centred molecules of biological interest through the construction of advanced magnetic circular dichroism (MCD) spectrometers. These facilities will be the best instruments of their kind, and will enable researchers at Australian institutions to enhance the quality of their research and remain ....New Generation Metalloenzyme Magnetic Circular Dichroism Spectrometer Systems. Funding is sought to enhance the existing collaborations between UQ, ANU, Sydney and other universities in the study of metal-centred molecules of biological interest through the construction of advanced magnetic circular dichroism (MCD) spectrometers. These facilities will be the best instruments of their kind, and will enable researchers at Australian institutions to enhance the quality of their research and remain internationally competitive through the application of modern MCD spectroscopic techniques to the study of metal-centred biomolecules. These facilities will drive a number of programs in the area of metalloenzyme and photosystem II research.Read moreRead less
Functional evolution and therapeutic potential of snake venom coagulotoxins. This project aims to identify and understand the factors that influence the useful function of key residues (parts of larger compounds) in Australian snake venom coagulotoxins, which alter blood-clotting ability. In recent years, snake venom compounds have been demonstrated as useful models from which to synthesise therapeutic drugs to improve health and well-being. This project will test these important toxins on model ....Functional evolution and therapeutic potential of snake venom coagulotoxins. This project aims to identify and understand the factors that influence the useful function of key residues (parts of larger compounds) in Australian snake venom coagulotoxins, which alter blood-clotting ability. In recent years, snake venom compounds have been demonstrated as useful models from which to synthesise therapeutic drugs to improve health and well-being. This project will test these important toxins on model systems that represent natural prey items in order to determine the molecular and functional evolution of blood-clot forming enzymes. Expected outcomes include substantial contributions to the body of evolutionary biology knowledge, as well as narrowing the search for the ultimate drug candidates.Read moreRead less
Genetic Basis of Variable Expression of Glycan Xeno-Autoantigens by Cattle. Meat and dairy products from cattle contain sugar structures (glycans) that are not made by humans. These structures can be recognised by the immune system and lead to allergic reactions, inflammation and potentially cancer. These non-human structures are called xeno-autoantigens or XAs. We have discovered individual cattle that do not produce one of these XAs. We will study the gene required to make XA in the XA-free ca ....Genetic Basis of Variable Expression of Glycan Xeno-Autoantigens by Cattle. Meat and dairy products from cattle contain sugar structures (glycans) that are not made by humans. These structures can be recognised by the immune system and lead to allergic reactions, inflammation and potentially cancer. These non-human structures are called xeno-autoantigens or XAs. We have discovered individual cattle that do not produce one of these XAs. We will study the gene required to make XA in the XA-free cattle to find the underlying mutation. The same approach will be used to look for natural XA-free individuals in other food species. This knowledge may enable us to create a test to facilitate the natural breeding of non-GMO, XA-free livestock to benefit Australian primary producers and provide safer food for consumers.Read moreRead less
MOLECULAR BREEDING OF CYTOCHROME P450 ENZYMES. Cytochrome P450s are enzymes that catalyse an impressive array of oxidative transformations. However, there is little available data on how to modify their substrate specificity and generate tailored biocatalysts. We plan to use an emerging technology known as DNA shuffling to create libraries of P450s with varying activities. These will then be screened for enzymes that can catalyse the formation of indigo (a blue dye) and indirubin (a chemother ....MOLECULAR BREEDING OF CYTOCHROME P450 ENZYMES. Cytochrome P450s are enzymes that catalyse an impressive array of oxidative transformations. However, there is little available data on how to modify their substrate specificity and generate tailored biocatalysts. We plan to use an emerging technology known as DNA shuffling to create libraries of P450s with varying activities. These will then be screened for enzymes that can catalyse the formation of indigo (a blue dye) and indirubin (a chemotherapeutic agent). The enzymes that catalyse indigo formation will be useful in the production of coloured transgenic plants and those that produce indirubin will have a role in gene therapy.Read moreRead less
Enzyme Electrochemical Communication. The ways that redox enzymes communicate with an electrochemical electrode are poorly understood and most systems rely on small molecule mediators as electron shuttles to complete the circuit. The few examples where direct (unmediated) enzyme electrochemistry has been achieved have relied on empirical experimental approaches in electrode modification. In this project a rational approach will be taken, starting with a mediated enzyme electrochemical system whi ....Enzyme Electrochemical Communication. The ways that redox enzymes communicate with an electrochemical electrode are poorly understood and most systems rely on small molecule mediators as electron shuttles to complete the circuit. The few examples where direct (unmediated) enzyme electrochemistry has been achieved have relied on empirical experimental approaches in electrode modification. In this project a rational approach will be taken, starting with a mediated enzyme electrochemical system which is then systematically deconstructed to produce a minimal enzyme-electrode that is stabilised by non-covalent forces and functions without a mediator. This rational approach will provide new routes to the direct enzyme electrochemistry of other enzyme systems as yet unexplored.Read moreRead less
Monolayer crystallization of membrane proteins. Membrane proteins comprise 25-40% of all proteins and conduct a myriad of finely tuned reactions in every cell. Despite their importance and diversity only ~40 membrane protein structures have been solved, due to the difficulty of producing high quality 2D and 3D crystals. We propose to develop and use the new monolayer crystallization technique, which employs a lipid monolayer as a crystallization template for 2D crystal production. A number of ....Monolayer crystallization of membrane proteins. Membrane proteins comprise 25-40% of all proteins and conduct a myriad of finely tuned reactions in every cell. Despite their importance and diversity only ~40 membrane protein structures have been solved, due to the difficulty of producing high quality 2D and 3D crystals. We propose to develop and use the new monolayer crystallization technique, which employs a lipid monolayer as a crystallization template for 2D crystal production. A number of important membrane proteins are available for these structural studies including ABC transporters, Caveolin-3 and the NS1 protein of Dengue virus, all of which are difficult to crystallize using conventional techniques.Read moreRead less