ORCID Profile
0000-0002-9739-6157
Current Organisation
Monash University
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Characterisation of Biological Macromolecules | Medicinal and Biomolecular Chemistry | Proteins and Peptides | Structural Biology (incl. Macromolecular Modelling) | Enzymes | Organic Chemical Synthesis | Biochemistry and Cell Biology
Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Biological Sciences | Human Pharmaceutical Treatments (e.g. Antibiotics) | Control of Animal Pests, Diseases and Exotic Species in Farmland, Arable Cropland and Permanent Cropland Environments |
Publisher: Wiley
Date: 25-08-2020
Abstract: The glycopeptide antibiotics (GPAs) are a fascinating ex le of complex natural product biosynthesis, with the nonribosomal synthesis of the peptide core coupled to a cytochrome P450‐mediated cyclisation cascade that crosslinks aromatic side chains within this peptide. Given that the challenges associated with the synthesis of GPAs stems from their highly crosslinked structure, there is great interest in understanding how biosynthesis accomplishes this challenging set of transformations. In this regard, the use of in vitro experiments has delivered important insights into this process, including the identification of the unique role of the X‐domain as a platform for P450 recruitment. In this minireview, we present an analysis of the results of in vitro studies into the GPA cyclisation cascade that have demonstrated both the tolerances and limitations of this process for modified substrates, and in turn developed rules for the future reengineering of this important antibiotic class.
Publisher: Oxford University Press (OUP)
Date: 2011
DOI: 10.1039/C0MT00081G
Abstract: Enzymes of the cytochrome P450 superfamily can catalyse many types of oxidative transformations of a erse substrate range. This review summarises recent work on an subset of P450s that accept their substrates in carrier protein-bound form. These ex les show how the oxidative power and precision that P450s bring to natural products biosynthesis can be coupled with the advantages of using a carrier protein as a scaffold for oxidation.
Publisher: Wiley
Date: 20-07-2016
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D3CC01578E
Abstract: Chemical stabilisation of carrier protein bound substrates in non-ribosomal peptide synthesis can result in a loss in activity of neighbouring catalytic domains.
Publisher: American Chemical Society (ACS)
Date: 14-04-2014
DOI: 10.1021/OL500840F
Abstract: A rapid protocol based on Fmoc-chemistry for the solid phase peptide synthesis of vancomycin- and teicoplanin-type peptides is described. Epimerization of highly racemization-prone arlyglycine derivatives is suppressed through optimized Fmoc-deprotection and coupling conditions. Starting from easily accessible Fmoc-protected amino acids, this strategy enables the enantioselective synthesis of peptides corresponding to intermediates found in vancomycin and teicoplanin biosynthesis with excellent purity and in high yields (38%-71%).
Publisher: Wiley
Date: 29-10-2012
Abstract: The products of cytochrome P450(BM3)-catalysed oxidation of cyclopropyl-containing dodecanoic acids are consistent with the presence of a cationic reaction intermediate, which results in efficient dehydrogenation of the rearranged probes by the enzyme. These results highlight the importance of enzyme-substrate complementarity, with a cationic intermediate occurring only when the probes used begin to erge from ideal substrates for this enzyme. This also aids in reconciling literature reports supporting the presence of cationic intermediates with certain cytochrome P450 enzyme/substrate pairs.
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D0CC07421G
Abstract: Chemical probes were used to intercept peptide biosynthetic intermediates in vancomycin formation in vivo .
Publisher: Elsevier BV
Date: 03-2011
DOI: 10.1016/J.ABB.2010.10.017
Abstract: Teicoplanin is a glycopeptide antibiotic with activity against Gram-positive bacteria and remains one of the last lines of clinical defense against certain bacterial infections. We have cloned, expressed, and purified the cytochrome P450 OxyE (CYP165D3) from the teicoplanin biosynthetic gene cluster of Actinoplanes teichomyceticus, which is responsible for the phenolic coupling of the aromatic side chains of the first and third peptide residues in the teicoplanin peptide. The crystal structure of OxyE has been determined to 2.5Å resolution, revealing the probable binding surface for the carrier protein substrate and an extension of the active site into a pocket located above the β-1 sheet. The binding of potential substrates to OxyE shows that peptidyl carrier protein-bound linear peptides bind to OxyE, albeit with low affinity in the absence of a phenolic cross-link that should normally be installed by another Oxy protein in the teicoplanin biosynthetic pathway. This result indicates that the carrier protein alone is not sufficient for tight substrate binding to OxyE and that the Oxy proteins sense the structure of the bound peptide in addition to the presence of the carrier protein, a feature distinct from other carrier protein/P450 systems.
Publisher: Royal Society of Chemistry (RSC)
Date: 2004
DOI: 10.1039/B315911F
Abstract: Novel cyclopropyl containing fatty acids are good substrates for P450(BM3) catalysed hydroxylation and analysis of their oxidation products indicates the presence of a radical intermediate (maximum rebound rate 2.6 x 10(10) s(-1)) and the absence of any cationic intermediate.
Publisher: Wiley
Date: 11-03-2026
Publisher: American Chemical Society (ACS)
Date: 30-04-2018
Abstract: Natural products such as the glycopeptide antibiotics (GPAs, including vancomycin and teicoplanin) are of great pharmaceutical importance due to their use against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus. GPAs are assembled in a complex process based on nonribosomal peptide synthesis and late-stage, multistep cross-linking of the linear heptapeptide performed by cytochrome P450 monooxygenases. These P450 enzymes demonstrate varying degrees of substrate selectivity toward the linear peptide precursor, with limited information available about their tolerance regarding modifications to amino acid residues within the essential antibiotic core of the GPA. In order to test the acceptance of altered residues by the P450-catalyzed cyclization cascade, we have explored the use of β-amino acids in both variable and highly conserved positions within GPA peptides. Our results indicate that the incorporation of β-amino acids at the C-terminus of the peptide leads to a dramatic reduction in the efficiency of peptide cyclization by the P450s during GPA biosynthesis, whereas replacement of residue 3 is well tolerated by the same enzymes. These results show that maintaining the C-terminal 3,5-dihydroxyphenylglycine residue is of key importance to maintain the efficiency of this complex and essential enzymatic cross-linking process.
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C6MB00675B
Abstract: Highlighting recent important discoveries from the biosynthesis of peptides by linear non-ribosomal peptide synthetase (NRPS) machineries.
Publisher: Elsevier BV
Date: 08-2023
Publisher: Wiley
Date: 17-01-2008
Abstract: The hydroxylation activity of the Thr268Ala mutant of P450(BM3) has been shown to occur to varying degrees with small alterations in the structure of a fatty-acid substrate. Ten substrates were investigated, including straight chain, branched chain and cis-cyclopropyl substituted fatty acids with a straight-chain length that varied between 12 and 16 carbon atoms. The efficacy of the hydroxylation activity appeared to be governed by the chain length of the substrate. Substrates possessing 14 to 15 carbons afforded the highest levels of activity, which were comparable with the wild-type enzyme. Outside of this window, straight-chain fatty acids showed reduced activity over the other substrate types. These results provide a cautionary tale concerning the loss of ferryl activity in such cytochrome P450 threonine to alanine mutants, as the nature of the substrate can determine the extent to which hydroxylation chemistry is abolished.
Publisher: American Chemical Society (ACS)
Date: 14-08-2020
Publisher: American Physical Society (APS)
Date: 30-01-2013
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7RA06518C
Abstract: P450 monooxygenase enzyme ComJ catalyzed biaryl ether bond formation with high efficiency and low stereoselectivity on selected complestatin-like peptide substrates.
Publisher: Wiley
Date: 03-08-2022
Abstract: We report our investigation of the utility of peptide crosslinking cytochrome P450 enzymes from biarylitide biosynthesis to generate a range of cyclic tripeptides from simple synthons. The crosslinked tripeptides produced by this P450 include both tyrosine-histidine (A-N-B) and tyrosine-tryptophan (A-O-B) crosslinked tripeptides, the latter a rare ex le of a phenolic crosslink to an indole moiety. Tripeptides are easily isolated following proteolytic removal of the leader peptide and can incorporate a wide range of amino acids in the residue inside the crosslinked tripeptide. Given the utility of peptide crosslinks in important natural products and the synthetic challenge that these can represent, P450 enzymes have the potential to play roles as important tools in the generation of high-value cyclic tripeptides for incorporation in synthesis, which can be yet further ersified using selective chemical techniques through specific handles contained within these tripeptides.
Publisher: American Society for Microbiology
Date: 30-06-2020
DOI: 10.1128/MSYSTEMS.00389-20
Abstract: Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F 420 , and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F 420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F 420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F 420 , determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F 420 .
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C7NP00063D
Abstract: This review highlights the scope of chemical transformations that cytochrome P450 enzymes catalyse within bacterial secondary metabolism.
Publisher: Elsevier BV
Date: 06-2005
Publisher: Wiley
Date: 02-2016
Abstract: Cyclization of glycopeptide antibiotic precursors occurs in either three or four steps catalyzed by Cytochrome P450 enzymes. Three of these enzymes have been structurally characterized to date with the second enzyme along the pathway, OxyA, escaping structural analysis. We are now able to present the structure of OxyAtei involved in teicoplanin biosynthesis - the same enzyme recently shown to be the first active OxyA homolog. In spite of the hydrophobic character of the teicoplanin precursor, the polar active site of OxyAtei and its affinity for certain azole inhibitors hint at its preference for substrates with polar decorations.
Publisher: Springer Science and Business Media LLC
Date: 09-02-2015
DOI: 10.1038/NATURE14141
Abstract: Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450-X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro--in particular the installation of the second crosslink in GPA biosynthesis--occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.
Publisher: Springer Science and Business Media LLC
Date: 03-02-2021
DOI: 10.1038/S41586-021-03195-X
Abstract: Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture
Publisher: Elsevier BV
Date: 12-2019
Publisher: Elsevier BV
Date: 12-2016
DOI: 10.1016/J.SBI.2016.05.018
Abstract: The importance of Cytochrome P450-catalyzed modifications of natural products produced by non-ribosomal peptide synthetase machineries is most apparent during glycopeptide antibiotic biosynthesis: specifically, the formation of essential amino acid side chains crosslinks in the peptide backbone of these clinically relevant antibiotics. These cyclization reactions take place whilst the peptide substrate remains bound to the non-ribosomal peptide synthetase in a process mediated by a conserved domain of previously unknown function-the X-domain. This review addresses recent advances in understanding P450 recruitment to non-ribosomal peptide synthetase-bound substrates and highlights the importance of both carrier proteins and the X-domain in different P450-catalyzed reactions.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6MB00373G
Abstract: Cytochrome P450 StaH catalyses the first cyclisation reaction in the biosynthesis of the glycopeptide antibiotic A47934 via recruitment to the NRPS machinery through interaction with the X-domain.
Publisher: Wiley
Date: 30-05-2020
DOI: 10.1111/FEBS.15350
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D0NA01018A
Abstract: Peptides containing vancomycin were able to self-assemble into antimicrobial 2D scaffolds.
Publisher: American Chemical Society (ACS)
Date: 11-10-2019
DOI: 10.1021/ACS.ORGLETT.9B03245
Abstract: Natural products are the greatest source of antimicrobial agents, although their structural complexity often renders synthetic production and ersification of key classes impractical. One pertinent ex le is the glycopeptide antibiotics (GPAs), which are highly challenging to synthesize due to their heavily cross-linked structures. Here, we report an optimized method that generates >75% tricyclic peptides from synthetic precursors in order to explore the acceptance of novel GPA precursor peptides by these key existent biosynthetic enzymes.
Publisher: Wiley
Date: 18-01-2023
Abstract: The glycopeptide antibiotics (GPAs) are a clinically approved class of antimicrobial agents that classically function through the inhibition of bacterial cell‐wall biosynthesis by sequestration of the precursor lipid II. The oxidative crosslinking of the core peptide by cytochrome P450 (Oxy) enzymes during GPA biosynthesis is both essential to their function and the source of their synthetic challenge. Thus, understanding the activity and selectivity of these Oxy enzymes is of key importance for the future engineering of this important compound class. Recent reports of GPAs that display an alternative mode of action and a wider range of core peptide structures compared to classic lipid II‐binding GPAs raises the question of the tolerance of Oxy enzymes for larger changes in their peptide substrates. In this work, we explore the ability of Oxy enzymes from the biosynthesis pathways of lipid II‐binding GPAs to accept altered peptide substrates based on a vancomycin template. Our results show that Oxy enzymes are more tolerant of changes at the N terminus of their substrates, whilst C‐terminal extension of the peptide substrates is deleterious to the activity of all Oxy enzymes. Thus, future studies should prioritise the study of Oxy enzymes from atypical GPA biosynthesis pathways bearing C‐terminal peptide extension to increase the substrate scope of these important cyclisation enzymes.
Publisher: Wiley
Date: 25-07-2022
Abstract: Detection of pyrophosphate is important in quantifying enzyme activity, particularly adenylation domain activity during non‐ribosomal peptide synthesis. The previous development of an enzyme coupled PP i /NADH assay allowed the measurement of such activity in an online fashion using commercially available components. Now, with a key enzyme ‐ 6‐phosphofructokinase ‐ no longer available, we have screened and identified viable replacement enzymes that can be expressed in high yield and that are far superior in activity to the now discontinued commercial product. This will support the ability of groups to continue to use this established online assay for pyrophosphate detection.
Publisher: American Chemical Society (ACS)
Date: 27-11-2019
Abstract: The glycopeptide antibiotics (GPAs) serve as an important ex le of the interplay of two powerful enzymatic classes in secondary metabolism: the coupling of nonribosomal peptide synthesis with oxidative aromatic cross-linking performed by cytochrome P450 enzymes. This interplay is responsible for the generation of the highly cross-linked peptide aglycone at the core of this compound class that is required for antibiotic activity and, as such, serves as an important point for the exploration of chemoenzymatic routes to understand the selectivity and mechanism of this complex cascade. Here, we demonstrate the effective reconstitution of enzymatic tetracyclization of synthetic teicoplanin-derived heptapeptides and furthermore discern the importance of the OxyE enzyme in maintaining effective cyclization of such peptides bearing 3,5-dihydroxyphenylglycine residues at position 3 in their structures. These results demonstrate the value of chemically synthesized probes for the elucidation of the enzyme mechanism underpinning the complex process of GPA cyclization and furthermore show the utility of the technique for probing the cyclization of non-natural GPA peptides by these powerful biosynthetic enzymes.
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D3SC01944F
Publisher: American Chemical Society (ACS)
Date: 27-11-2019
Publisher: Wiley
Date: 20-07-2016
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8NP00038G
Abstract: Non-ribosomal peptide synthetase (NRPS) machineries are complex, multi-domain proteins that are responsible for the biosynthesis of many important, peptide-derived compounds. In this review, we present the current state of understanding of the protein–protein interactions that govern NRPS-mediated biosynthesis.
Publisher: Cold Spring Harbor Laboratory
Date: 03-05-2020
DOI: 10.1101/2020.05.02.073981
Abstract: Actinobacteria produce numerous antibiotics and other specialised metabolites with important applications in medicine and agriculture. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR. Here, we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding by single particle cryo-electron microscopy of an MmfR-operator complex. Electrophoretic mobility shift assays with MmfR mutants and synthetic AHFCA analogues illuminate the role played by in idual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of Actinobacterial hormones and their associated TFTRs in synthetic biology and novel antibiotic discovery.
Publisher: Cold Spring Harbor Laboratory
Date: 11-02-2023
DOI: 10.1101/2023.02.10.526856
Abstract: Antibiotics have been an essential part of modern medicine since their initial discovery. The continuous search for new antibiotic candidates remains a necessity given the increasing emergence of resistance to antimicrobial compounds among pathogens. The glycopeptide antibiotics (GPAs) represent an important group of last resort antibiotics which inhibit bacterial growth through non-covalent binding to the cell wall precursor lipid II. The so far reported GPAs exhibit an enormous ersity in the biosynthetic gene clusters that encode their production, which is in turn reflected in the variety of their structures. GPAs are typically composed of seven amino acids, which are highly crosslinked and decorated with a variable collection of sugar moieties as well as other modifications. Based on their structural characteristics, they have been classified into four main types. More recently, atypical GPAs have been identified that differ from type I-IV GPAs in both their structure and function and have consequently been classified as type V GPAs. Given these differences, we studied the phylogeny of all gene sequences related to the biosynthesis of the GPAs and observed a clear evolutionary ersification between the lipid II binding GPA classes and the so-called type V GPAs. Here we suggest the adoption of a phylogeny-driven reclassification and a separation of classical lipid II binding GPAs from type V GPAs, which we propose to identify instead as glycopeptide- related peptides (GRPs).
Publisher: Royal Society of Chemistry (RSC)
Date: 2004
DOI: 10.1039/B311652B
Abstract: Cytochrome p450(BioI)(CYP107H1) is believed to supply pimelic acid equivalents for biotin biosynthesis in Bacillus subtilis: we report here that the mechanistic pathway adopted by this multifunctional p450 for the in-chain cleavage of fatty acids is via consecutive formation of alcohol and threo-diol intermediates, with the likely absolute configuration of the intermediates also reported.
Publisher: Wiley
Date: 30-10-2014
Abstract: Bacterial cytochrome P450s form a remarkable clade of the P450 superfamily of oxidative hemoproteins, and are often involved in the biosynthesis of complex natural products. Those in a subgroup known as "Oxy enzymes" play a crucial role in the biosynthesis of glycopeptide antibiotics, including vancomycin and teicoplanin. The Oxy enzymes catalyze crosslinking of aromatic residues in the non-ribosomal antibiotic precursor peptide while it remains bound to the non-ribosomal peptide synthetase (NRPS) this crosslinking secures the three-dimensional structure of the glycopeptide, crucial for antibiotic activity. We have characterized OxyBtei , the first of the Oxy enzymes in teicoplanin biosynthesis. Our results reveal that OxyBtei possesses a structure similar to those of other Oxy proteins and is active in crosslinking NRPS-bound peptide substrates. However, OxyBtei displays a significantly altered activity spectrum against peptide substrates compared to its well-studied vancomycin homologue.
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D2NP00044J
Abstract: Streptomyces are ubiquitous in terrestrial and marine environments, where they display a fascinating metabolic ersity.
Publisher: Wiley
Date: 09-07-2014
Abstract: Studying the interplay between nonribosomal peptide synthetases (NRPS), a major source of secondary metabolites, and crucial external modifying enzymes is a challenging task since the interactions involved are often transient in nature. By applying a range of synthetic inhibitor-type compounds, a stabilized complex appropriate for structural analysis was generated for such a tailoring enzyme and an NRPS domain. The complex studied comprises an NRPS peptidyl carrier protein (PCP) domain bound to the Cytochrome P450 enzyme that is crucial for the provision of β-hydroxylated amino acid precursors in the biosynthesis of the cyclic depsipeptide skyllamycin. The structure reveals that complex formation is governed by hydrophobic interactions, the presence of which can be controlled through minor alterations in PCP structure that enable selectivity amongst multiple highly similar PCP domains.
Publisher: American Chemical Society (ACS)
Date: 16-08-2021
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C4MB00665H
Abstract: The structure of CYP109B1 from Bacillus subtilis , which catalyses the oxidation of ionones, has been determined. This will allow the future design of more efficient biocatalytic monooxygenase systems.
Publisher: Elsevier BV
Date: 08-2010
Publisher: American Chemical Society (ACS)
Date: 15-08-2003
DOI: 10.1021/OL035254E
Abstract: [reaction: see text] Oxidation of tetradecanoic and hexadecanoic acids by cytochrome P450(BioI) (CYP107H1) produces mainly the 11-, 12-, and 13-hydroxy C(14) fatty acids and the 11- to 15-hydroxy C(16) fatty acids, respectively. In contrast to previous reports, terminal hydroxylation is not observed. The enantiospecificity of fatty acid hydroxylation by P450(BioI) was also determined, and the enzyme was shown to be moderately selective for production of the (R)-alcohols.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6CC06975D
Abstract: Remarkable promiscuity of N -methyltransferases enables modulation of biological activity as well as bio-orthogonal labelling of glycopeptide antibiotics and biosynthetic intermediates.
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5NP00025D
Abstract: Phenylglycine-type amino acids occur in a wide variety of peptide natural products. Herein structures and properties of these peptides as well as the biosynthetic origin and incorporation of phenylglycines are discussed.
Publisher: Wiley
Date: 11-2006
DOI: 10.1111/J.1471-4159.2006.04286.X
Abstract: Neuronal communication relies on the fusion of neurotransmitter-containing vesicles with the neuronal plasma membrane. Recent genetic studies have highlighted the critical role played by polyunsaturated fatty acids in neurotransmission, however, there is little information available about which fatty acids act on exocytosis and, more importantly, by what mechanism. We have used permeabilized chromaffin cells to screen various fatty acids of the n-3 and n-6 series for their acute effects on exocytosis. We have demonstrated that an n-6 series polyunsaturated fatty acid, arachidonic acid, potentiates secretion from intact neurosecretory cells regardless of the secretagogue used. We have shown that arachidonic acid dose dependently increases soluble NSF attachment protein receptor complex formation in chromaffin cells and bovine cortical brain extracts and that a non-hydrolysable analogue of arachidonic acid causes a similar increase in SNARE complex formation. This prompted us to examine the effect of arachidonic acid on SNARE protein interactions with Munc18a, a protein known to prevent Syntaxin1a engagement into the SNARE complex in vitro. In the presence of arachidonic acid, we show that Munc18a can interact with the neuronal SNARE complex in a dose-dependent manner. We further demonstrate that arachidonic acid directly interacts with Syntaxin1a.
Publisher: American Chemical Society (ACS)
Date: 26-02-2005
DOI: 10.1021/JO047985D
Abstract: [reaction: see text] The mechanism of aliphatic hydroxylation by cytochromes P450 has been the subject of intense debate with several proposed mechanistic alternatives. Various cyclopropyl containing compounds (radical clocks), which can produce both unrearranged and ring opened products upon oxidation, have been key tools in these investigations. In this study, we introduce several cyclopropyl containing fatty acids 1a-4a with which to probe the mechanism of P450s capable of fatty acid hydroxylation. The probes are shown to be capable of distinguishing radical from cationic intermediates due to the rapid equilibration of isomeric cyclopropyl cations. Ring opening of a radical intermediate in an oxidative transformation is expected to yield a single rearranged alcohol, whereas a cation isomerizes prior to ring opening, leading to two isomeric homoallylic alcohols. Oxidation of these probes by P450(BM3) and P450(BioI) gives results consistent with a radical but not a cationic intermediate in fatty acid hydroxylation by these enzymes. Quantitation of the unrearranged and ring opened products gives remarkably homogeneous rates for oxygen rebound of (2-3) x 10(10) s(-1). The effects of introduction of a cyclopropane ring into a fatty acid upon the regiochemistry of hydroxylation are discussed.
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C9SC03678D
Abstract: A complex interplay of non-ribosomal peptide synthetase domains works together with trans -acting enzymes to ensure effective GPA biosynthesis.
Publisher: Wiley
Date: 23-02-2016
Abstract: Nonribosomal peptide synthetases (NRPSs) produce many important and structurally complex natural products. Because of their architectures, reprogramming NRPSs has long been attempted to access new bioactive compounds. However, detailed characterization of NRPS catalysis and substrate selectivity by adenylation (A) domains is needed to support such efforts. We present a simple coupled NADH yrophosphate (PPi ) detection assay for analyzing A domain catalysis in vitro. PPi formation is coupled to the consumption of NADH by four enzymatic steps and is detected spectroscopically (λ=340 nm) for simple analysis. We demonstrate the effectiveness of this assay with several adenylation domains, including a stand-alone A domain (DltA, cell wall biosynthesis) and an embedded A domain (Tcp10, teicoplanin biosynthesis). Substrate acceptance of the Tcp10 A domain was explored for the first time, thus demonstrating the applicability of the assay for complex, multi-domain NRPSs.
Publisher: Royal Society of Chemistry (RSC)
Date: 2006
DOI: 10.1039/B601202G
Abstract: Branched chain fatty acids are substrates for cytochrome P450(BM3) (CYP102) from Bacillus megaterium oxidation of C15 and C17 iso and anteiso fatty acids by P450(BM3) leads to the formation of hydroxylated products that possess high levels of regiochemical and stereochemical purity.
Publisher: Springer Science and Business Media LLC
Date: 25-10-2021
DOI: 10.1038/S41467-021-26244-5
Abstract: The pathogen Staphylococcus aureus can readily develop antibiotic resistance and evade the human immune system, which is associated with reduced levels of neutrophil recruitment. Here, we present a class of antibacterial peptides with potential to act both as antibiotics and as neutrophil chemoattractants. The compounds, which we term ‘antibiotic-chemoattractants’, consist of a formylated peptide (known to act as chemoattractant for neutrophil recruitment) that is covalently linked to the antibiotic vancomycin (known to bind to the bacterial cell wall). We use a combination of in vitro assays, cellular assays, infection-on-a-chip and in vivo mouse models to show that the compounds improve the recruitment, engulfment and killing of S. aureus by neutrophils. Furthermore, optimizing the formyl peptide sequence can enhance neutrophil activity through differential activation of formyl peptide receptors. Thus, we propose antibiotic-chemoattractants as an alternate approach for antibiotic development.
Publisher: Elsevier BV
Date: 12-2019
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C4OB02452D
Abstract: One Cytochrome P450 enzyme performs the phenolic crosslinking of a range of chemically synthesized, carrier-protein loaded glycopeptide antibiotic precursor peptides.
Publisher: Wiley
Date: 18-01-2023
Abstract: A highly effective 2‐step system for site‐specific antibody modification and conjugation of the monoclonal antibody Herceptin (commercially available under Trastuzumab) in a cysteine‐independent manner was used to generate labelled antibodies for in vivo imaging. The first step contains redox‐activated chemical tagging (ReACT) of thioethers via engineered methionine residues to introduce specific alkyne moieties, thereby offering a novel easy way to fundamentally change the process of antibody bioconjugation. The second step involves modification of the introduced alkyne via azide‐alkyne cycloaddition ′click′ conjugation. The versatility of this 2‐step approach is demonstrated here by the selective incorporation of a fluorescent dye but can also be applied to a wide variety of different conjugation partners depending on the desired application in a facile manner. Methionine‐modified antibodies were characterised in vitro, and the diagnostic potential of the most promising variant was further analysed in an in vivo xenograft animal model using a fluorescence imaging modality. This study demonstrates how methionine‐mediated antibody conjugation offers an orthogonal and versatile route to the generation of tailored antibody conjugates with in vivo applicability.
Publisher: Wiley
Date: 12-12-2008
Publisher: American Chemical Society (ACS)
Date: 06-08-2010
DOI: 10.1021/BI100910Y
Abstract: Cytochrome P450 CypX (CYP134A1), isolated from Bacillus subtilis, has previously been implicated in the three-step oxidative transformation of the diketopiperazine cyclo-l-leucyl-l-leucyl into pulcherriminic acid, a precursor of the extracellular iron chelate pulcherrimin. In this study, we present the first experimental data relating to CYP134A1, where we show that CYP134A1 binds cyclo-l-leucyl-l-leucyl with an affinity of 24.5 +/- 0.5 muM. Structurally related diketopiperazines sharing similar alkyl side chains to cyclo-l-leucyl-l-leucyl also bind to CYP134A1 with comparable affinity. CYP134A1 is capable of catalyzing the in vitro oxidation of diketopiperazine substrates when supported with several alternate electron transfer partner systems. Products containing one additional oxygen atom and which are intermediate products of the expected pulcherriminic acid were identified by GCMS. The oxidation of related diketopiperazines reveals that different oxidative pathways exist for CYP134A1-catalyzed diketopiperazine oxidation. The crystal structure of CYP134A1 has been determined to 2.7 A resolution in the absence of substrate and in the presence of bound phenylimidazole ligands to 3.1 and 3.2 A resolution. The active site is dominated by hydrophobic residues and contains an unusual proline residue in place of the normally conserved alcohol residue that typically plays an important role in oxygen activation. The B-B(2) substrate recognition loop, which forms part of the active site, shows considerable flexibility and was found in both open and closed conformations in different structures. These results represent the first insights into the structural and biochemical basis underlying the multistep oxidation catalyzed by CYP134A1.
Publisher: Wiley
Date: 05-02-2015
DOI: 10.1002/PROT.24758
Abstract: The biosynthesis of the glycopeptide antibiotics, of which teicoplanin and vancomycin are representative members, relies on the combination of non-ribosomal peptide synthesis and modification of the peptide by cytochrome P450 (Oxy) enzymes while the peptide remains bound to the peptide synthesis machinery. We have structurally characterized the final peptidyl carrier protein domain of the teicoplanin non-ribosomal peptide synthetase machinery: this domain is believed to mediate the interactions with tailoring Oxy enzymes in addition to its function as a shuttle for intermediates between multiple non-ribosomal peptide synthetase domains. Using solution state NMR, we have determined structures of this PCP domain in two states, the apo and the post-translationally modified holo state, both of which conform to a four-helix bundle assembly. The structures exhibit the same general fold as the majority of known carrier protein structures, in spite of the complex biosynthetic role that PCP domains from the final non-ribosomal peptide synthetase module must play in glycopeptide antibiotic biosynthesis. These structures thus support the hypothesis that it is subtle rearrangements, rather than dramatic conformational changes, which govern carrier protein interactions and selectivity during non-ribosomal peptide synthesis.
Publisher: American Chemical Society (ACS)
Date: 30-09-2013
DOI: 10.1021/CB400555E
Abstract: The generation of modified amino acid precursors for incorporation in nonribosomal peptide synthesis (NRPS) plays a crucial, if often understated, role in the generation of peptide natural products. The biosynthesis of the cyclic depsipeptide skyllamycin requires three β-hydroxylated amino acid precursors, with in vivo gene inactivation experiments implicating cytochrome P450sky (CYP163B3) in the hydroxylation of these amino acids. Here, we demonstrate the in vitro oxidation of l-amino acid substrates bound to peptidyl carrier protein (PCP) domains 5, 7, and 11 of the skyllamycin nonribosomal synthetase by P450sky. Selectivity for these domains over other PCP domains could be demonstrated, with hydroxylation selective for l-amino acids and stereospecific in nature resulting in the (2S,3S)-configuration. The oxidation of amino acids or small molecule substrate analogues was not supported, demonstrating the necessity of the carrier protein in P450sky-catalyzed hydroxylation. The binding of aminoacyl-PCP substrates to P450sky was detected for the catalytically active PCP7 but not for the catalytically inactive PCP10, indicating carrier protein-mediated selectivity in P450sky substrate binding. X-ray crystal structures of P450sky reveal a 3D-structure with a highly open active site, the size of which is dictated by the carrier protein bound nature of the substrate. P450sky is the first P450 demonstrated to not only interact directly with PCP-bound amino acids within the peptide-forming NRPS but also to do so with three different PCP domains in a specific fashion. This represents an expansion of the complexity and scope of NRPS-mediated peptide synthesis, with the generation of hydroxylated amino acid precursors occurring through the interaction of P450 enzymes following, rather than prior to, the selection of amino acids by NRPS-adenylation domains.
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C8SC03530J
Abstract: The condensation domain synthesising the last peptide bond in glycopeptide antibiotic biosynthesis has a preference for linear peptide substrates, with effective peptide formation linked to the rate of amino acid activation by the preceding adenylation domain.
Publisher: Portland Press Ltd.
Date: 26-07-2010
DOI: 10.1042/BST0380934
Abstract: The cytochromes P450 (P450s) are a superfamily of oxidative haemoproteins that are capable of catalysing a vast range of oxidative transformations, including the oxidation of unactivated alkanes, often with high stereo- and regio-selectivity. Fatty acid hydroxylation by P450s is widespread across both bacteria and higher organisms, with the sites of oxidation and specificity of oxidation varying from system to system. Several key ex les are discussed in the present article, with the focus on P450BioI (CYP107H1), a biosynthetic P450 found in the biotin operon of Bacillus subtilis. The biosynthetic function of P450BioI is the formation of pimelic acid, a biotin precursor, via a multiple-step oxidative cleavage of long-chain fatty acids. P450BioI is a member of an important subgroup of P450s that accept their substrates not free in solution, but rather presented by a separate carrier protein. Structural characterization of the P450BioI–ACP (acyl-carrier protein) complex has recently been performed, which has revealed the basis for the oxidation of the centre of the fatty acid chain. The P450BioI–ACP structure is the first such P450–carrier protein complex to be characterized structurally, with important implications for other biosynthetically intriguing P450–carrier protein complexes.
Publisher: Wiley
Date: 11-05-2020
Publisher: Elsevier BV
Date: 08-2007
Publisher: Wiley
Date: 04-12-2006
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C7CC09409D
Abstract: Non-ribosomal peptides contain an array of amino acid building blocks that can present challenges for the synthesis of important intermediates. Here we report a route to incorporate phenylglycine residues in peptide thioesters without significant racemisation.
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8NP90037J
Abstract: The Natural Product Reports themed issue on Understanding biosynthetic protein–protein interactions is introduced by the Guest Editors, David Ackerley, Gregory Challis and Max Cryle.
Publisher: American Chemical Society (ACS)
Date: 12-2017
DOI: 10.1021/ACSCHEMBIO.7B00943
Abstract: The biosynthesis of the glycopeptide antibiotics (GPAs)-which include teicoplanin and vancomycin-is a complex enzymatic process relying on the interplay of nonribosomal peptide synthesis and a cytochrome P450-mediated cyclization cascade. This unique cyclization cascade generates the highly cross-linked state of these nonribosomal peptides, which is crucial for their antimicrobial activity. Given that these essential oxidative transformations occur while the peptide remains bound to the terminal module of the nonribosomal peptide synthetase (NRPS) machinery, it is important to assess the selectivity of the terminal thioesterase (TE) domain and how this domain contributes to the maintenance of an efficient biosynthetic pathway while at the same time ensuring GPA maturation is completed. In this study, we report the in vitro characterization of the thioesterase domain from teicoplanin biosynthesis, the first GPA thioesterase to be characterized. Our results show that the activity of this TE domain relies on the presence of an unusual extended N-terminal linker region that appears to be unique to the NRPS machineries found in GPA biosynthesis. In addition, we show that the activity of this domain against carrier protein bound substrates is dramatically enhanced for mature GPA aglycones as opposed to linear peptides and partially cyclized intermediates. These results demonstrate how the interplay between NRPS and P450s during late stage GPA biosynthesis is not only maintained but also leads to the efficient production of mature GPA aglycones. Thus, GPA TE domains represent another impressive ex le of the ability of TE domains to act as logic gates during NRPS biosynthesis, ensuring that essential late-stage peptide modifications are completed before catalyzing the release of the mature, bioactive peptide product.
Publisher: Springer Science and Business Media LLC
Date: 04-05-2021
DOI: 10.1038/S41467-021-22623-0
Abstract: Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.
Publisher: Elsevier BV
Date: 12-2019
Publisher: Royal Society of Chemistry (RSC)
Date: 2020
DOI: 10.1039/D0SC03483E
Abstract: Redesign of the non-ribosomal peptide synthetase (NRPS) from teicoplanin biosynthesis has been extensively investigated via domain exchange, interface reengineering and through engineering communication between isolated NRPS modules.
Publisher: Proceedings of the National Academy of Sciences
Date: 14-10-2008
Abstract: Cytochrome P450 BioI (CYP107H1) from the biotin operon of Bacillus subtilis forms a seven-carbon diacid through a multistep oxidative cleavage of a fatty acid linked to acyl carrier protein (ACP). Crystal structures of P450 BioI in complex with three different length fatty acyl -ACP ( Escherichia coli ) ligands show that P450 BioI binds the fatty acid such as to force the carbon chain into a U-shape above the active site heme. This positions the C7 and C8 carbons for oxidation, with a large additional cavity extending beyond the heme to accommodate the methyl termini of fatty acids beyond the site of cleavage. The structures explain the experimentally observed lack of stereo- and regiospecificity in the hydroxylation and cleavage of free fatty acids. The P450 BioI -ACP complexes represent the only structurally characterized P450-carrier protein complexes to date, which has allowed the generation of a model of the interaction of the vancomycin biosynthetic P450 OxyB with its proposed carrier protein bound substrate.
Publisher: Wiley
Date: 03-08-2022
Abstract: We report our investigation of the utility of peptide crosslinking cytochrome P450 enzymes from biarylitide biosynthesis to generate a range of cyclic tripeptides from simple synthons. The crosslinked tripeptides produced by this P450 include both tyrosine‐histidine (A− N −B) and tyrosine‐tryptophan (A− O −B) crosslinked tripeptides, the latter a rare ex le of a phenolic crosslink to an indole moiety. Tripeptides are easily isolated following proteolytic removal of the leader peptide and can incorporate a wide range of amino acids in the residue inside the crosslinked tripeptide. Given the utility of peptide crosslinks in important natural products and the synthetic challenge that these can represent, P450 enzymes have the potential to play roles as important tools in the generation of high‐value cyclic tripeptides for incorporation in synthesis, which can be yet further ersified using selective chemical techniques through specific handles contained within these tripeptides.
Publisher: Proceedings of the National Academy of Sciences
Date: 14-07-2009
Abstract: Archae possess unique biochemical systems quite distinct from the pathways present in eukaryotes and eubacteria. 7,8-Dimethyl-8-hydroxy-5deazaflavin (F 0 ) and F 420 are unique deazaflavin-containing coenzyme and methanogenic signature molecules, essential for a variety of biochemical transformations associated with methane biosynthesis and light-dependent DNA repair. The deazaflavin cofactor system functions during methane biosynthesis as a low-potential hydrid shuttle F 420 /F 420 H 2 . In DNA photolyase repair proteins, the deazaflavin cofactor is in the deprotonated state active as a light-collecting energy transfer pigment. As such, it converts blue sunlight into energy used by the proteins to drive an essential repair process. Analysis of a eukaryotic (6-4) DNA photolyase from Drosophila melanogaster revealed a binding pocket, which tightly binds F 0 . Residues in the pocket activate the cofactor by deprotonation so that light absorption and energy transfer are switched on. The crystal structure of F 0 in complex with the D. melanogaster protein shows the atomic details of F 0 binding and activation, allowing characterization of the residues involved in F 0 activation. The results show that the F 0 /F 420 coenzyme system, so far believed to be strictly limited to the archael kingdom of life, is far more widespread than anticipated. Analysis of a D. melanogaster extract and of a DNA photolyase from the primitive eukaryote Ostreococcus tauri provided direct proof for the presence of the F 0 cofactor also in higher eukaryotes.
Publisher: Wiley
Date: 31-01-2023
Publisher: Springer Science and Business Media LLC
Date: 13-06-2019
DOI: 10.1038/S41467-019-10384-W
Abstract: Kistamicin is a ergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A- O -B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.
Publisher: American Chemical Society (ACS)
Date: 24-02-2017
DOI: 10.1021/ACS.BIOCHEM.6B01102
Abstract: The activity of glycopeptide antibiotics (GPAs) depends upon important structural modifications to their precursor heptapeptide backbone: specifically, the cytochrome P450-catalyzed oxidative cross-linking of aromatic side chains as well as the halogenation of specific residues within the peptide. The timing of halogenation and its effect on the cyclization of the peptide are currently unclear. Our results show that chlorination of peptide precursors improves their processing by P450 enzymes in vitro, which provides support for GPA halogenation occurring prior to peptide cyclization during nonribosomal peptide synthesis. We could also determine that the activity of the second enzyme in the oxidative cyclization cascade, OxyA, remains higher for chlorinated peptide substrates even when the biosynthetic GPA product possesses an altered chlorination pattern, which supports the role of the chlorine atoms in orienting the peptide substrate in the active site of these enzymes.
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7SC00460E
Abstract: Halogenase enzymes involved in glycopeptide antibiotic biosynthesis accept aminoacyl-carrier protein substrates.
Publisher: Wiley
Date: 02-12-2017
DOI: 10.1002/PSC.2943
Abstract: Understanding the structure and function of protein complexes and multi-domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein rotein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon Sortase-mediated and Native chemical ligation using synthetic Peptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps - sortase-mediated and native chemical ligation - together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Publisher: Research Square Platform LLC
Date: 06-01-2021
DOI: 10.21203/RS.3.RS-125509/V1
Abstract: Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report the first structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this previously uncharacterized complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.
Publisher: Proceedings of the National Academy of Sciences
Date: 31-01-2019
Abstract: The protein Ebony from Drosophila melanogaster plays a central role in the regulation of histamine and dopamine in various tissues through condensation of these amines with β-alanine. Ebony is a rare ex le of a nonribosomal peptide synthetase (NRPS) from a higher eukaryote and contains a C-terminal sequence that does not correspond to any previously characterized NRPS domain. We have structurally characterized this C-terminal domain and have discovered that it adopts the aryl-alkylamine- N -acetyl transferase (AANAT) fold, which is unprecedented in NRPS biology. Through analysis of ligand-bound structures, activity assays, and binding measurements, we have determined how this atypical condensation domain is able to provide selectivity for both the carrier protein-bound amino acid and the amine substrates, a situation that remains unclear for standard condensation domains identified to date from NRPS assembly lines. These results demonstrate that the C terminus of Ebony encodes a eukaryotic ex le of an alternative type of NRPS condensation domain they also illustrate how the catalytic components of such assembly lines are significantly more erse than a minimal set of conserved functional domains.
Start Date: 2018
End Date: 2021
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2019
End Date: 2021
Funder: Australian Research Council
View Funded ActivityStart Date: 2020
End Date: 2026
Funder: Australian Research Council
View Funded ActivityStart Date: 2024
End Date: 2027
Funder: Marsden Fund
View Funded ActivityStart Date: 2017
End Date: 2019
Funder: Australian Research Council
View Funded ActivityStart Date: 2017
End Date: 12-2019
Amount: $431,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2021
End Date: 12-2023
Amount: $360,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2019
End Date: 12-2021
Amount: $480,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 01-2021
End Date: 01-2028
Amount: $35,000,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 04-2017
End Date: 12-2017
Amount: $850,000.00
Funder: Australian Research Council
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