Siderophores as anchors in artificial metalloenzymes
Lead Research Organisation:
University of York
Department Name: Chemistry
Abstract
The project involves the development of artificial metalloenzymes that consist of a synthetic catalytic centre that is linked via an anchor group to a protein that acts as a scaffold. This design allows the combination of the chemical reactivity of organometallic catalysts with the selectivity and biocompatibility of proteins. Such artificial enzymes would allow existing catalytic processes to run under mild conditions and make new biotechnological transformations possible. In addition, such artificial enzymes could be used to activate antibiotics.
The anchor groups to be investigated in this project are based on siderophores, which are ligands that are produced by bacteria for the uptake of essential iron. The iron(III)-siderophore complexes formed are extremely stable and actively transported into the bacterial cell. In Gram-negative bacteria, transport involves recognition and uptake via a specific outer membrane receptor, followed by capture by a periplasmic binding protein and delivery to an inner membrane transporter for translocation into the cytoplasm.
It is the periplasmic siderophore binding proteins that will be explored as novel protein scaffolds in artificial enzymes. The advantage of this approach is the active transport of the synthetic catalysts into the bacterial cell via the siderophore uptake system. Once the catalysts are taken-up along with the iron siderophore complexes, they are captured by their respective binding proteins. Hence the target artificial enzymes self-assemble and accumulate in the periplasm of the bacterial cell, where they may be used for catalytic transformations or the activation of antimicrobial prodrugs. In this way, by taking advantage of the bacterial iron-uptake pathway that is mediated by siderophores, the metabolism of the bacterial cell is exploited to support chemical transformations in vivo.
The anchor groups to be investigated in this project are based on siderophores, which are ligands that are produced by bacteria for the uptake of essential iron. The iron(III)-siderophore complexes formed are extremely stable and actively transported into the bacterial cell. In Gram-negative bacteria, transport involves recognition and uptake via a specific outer membrane receptor, followed by capture by a periplasmic binding protein and delivery to an inner membrane transporter for translocation into the cytoplasm.
It is the periplasmic siderophore binding proteins that will be explored as novel protein scaffolds in artificial enzymes. The advantage of this approach is the active transport of the synthetic catalysts into the bacterial cell via the siderophore uptake system. Once the catalysts are taken-up along with the iron siderophore complexes, they are captured by their respective binding proteins. Hence the target artificial enzymes self-assemble and accumulate in the periplasm of the bacterial cell, where they may be used for catalytic transformations or the activation of antimicrobial prodrugs. In this way, by taking advantage of the bacterial iron-uptake pathway that is mediated by siderophores, the metabolism of the bacterial cell is exploited to support chemical transformations in vivo.
Planned Impact
In addition to the immediate benefit of advancing the development of artificial enzymes, the proposed work may lead to the development of new catalysts with the potential to improve the biocompatibility of industrially relevant processes. In addition, our approach may enable the development of catalysts for transformations that have no equivalent either in homogeneous or enzymatic catalysis or the running of existing catalytic processes in a more sustainable way. A particular strength of the proposed protein-based catalysts lies in the preparation of enantiomerically pure molecules.
Furthermore, the proposed work may lead to the development of novel antimicrobial prodrugs. With the current lack of industrial research in the area of antimicrobials and bacterial resistance on the rise, it is particularly important that academics contribute to the field. We anticipate that the insights generated and researchers trained through our research will strengthen antimicrobial research nationally and internationally and that our publications will help to inform policy-makers. In the long-term, the proposed research could improve the health and quality of life of patients, if successful. In particular prodrugs that delay the development of resistance would be highly beneficial, since drug resistance tends to prolong the duration of an infection.
The proposal is thus relevant to several areas identified as of strategic importance by the research councils, including manufacturing for the future, catalysis, frontier manufacturing, sustainable industrial systems and the EPSRC challenge theme healthcare technologies.
Hence, the work could have considerable economic and societal impact in the long-term. In order to ensure the commercial potential of promising catalysts or antimicrobials, we will seek both intellectual property (IP) protection and industrial expertise. Help will be provided by the University of York Enterprise and Innovation Office. Once IP protection has been obtained, the results obtained will be published as communications and as full papers. In addition, lectures and posters will be presented at conferences and during visits to research institutions.
Since the move towards greener chemical technology is of much current interest and importance, the proposed work provides an opportunity to engage with the public and to raise awareness of environmental and sustainability issues. In addition, we believe that it is important to inform the public better about the problem of antibiotic resistance. The PI has already published an article in Education in Chemistry on the use of siderophores in antimicrobials, given a public lecture on the subject, and talked at schools events. In addition, we have offered many undergraduate projects on antibiotic research over the past five years, which proved to be very successful. We intend to continue and extend these and related outreach activities further.
The work will result in the training of highly skilled PDRAs, who will be well prepared for employment in either the academic and industrial sector. In addition, new graduates and final year undergraduates working on related projects will benefit from the training provided.
Furthermore, the proposed work may lead to the development of novel antimicrobial prodrugs. With the current lack of industrial research in the area of antimicrobials and bacterial resistance on the rise, it is particularly important that academics contribute to the field. We anticipate that the insights generated and researchers trained through our research will strengthen antimicrobial research nationally and internationally and that our publications will help to inform policy-makers. In the long-term, the proposed research could improve the health and quality of life of patients, if successful. In particular prodrugs that delay the development of resistance would be highly beneficial, since drug resistance tends to prolong the duration of an infection.
The proposal is thus relevant to several areas identified as of strategic importance by the research councils, including manufacturing for the future, catalysis, frontier manufacturing, sustainable industrial systems and the EPSRC challenge theme healthcare technologies.
Hence, the work could have considerable economic and societal impact in the long-term. In order to ensure the commercial potential of promising catalysts or antimicrobials, we will seek both intellectual property (IP) protection and industrial expertise. Help will be provided by the University of York Enterprise and Innovation Office. Once IP protection has been obtained, the results obtained will be published as communications and as full papers. In addition, lectures and posters will be presented at conferences and during visits to research institutions.
Since the move towards greener chemical technology is of much current interest and importance, the proposed work provides an opportunity to engage with the public and to raise awareness of environmental and sustainability issues. In addition, we believe that it is important to inform the public better about the problem of antibiotic resistance. The PI has already published an article in Education in Chemistry on the use of siderophores in antimicrobials, given a public lecture on the subject, and talked at schools events. In addition, we have offered many undergraduate projects on antibiotic research over the past five years, which proved to be very successful. We intend to continue and extend these and related outreach activities further.
The work will result in the training of highly skilled PDRAs, who will be well prepared for employment in either the academic and industrial sector. In addition, new graduates and final year undergraduates working on related projects will benefit from the training provided.
Organisations
Publications
Baranska NG
(2022)
Electrochemical and Solution Structural Characterization of Fe(III) Azotochelin Complexes: Examining the Coordination Behavior of a Tetradentate Siderophore.
in Inorganic chemistry
Bilitewski U
(2017)
Chemical and Biological Aspects of Nutritional Immunity-Perspectives for New Anti-Infectives that Target Iron Uptake Systems.
in Angewandte Chemie (International ed. in English)
Black CM
(2022)
Synthesis and antimicrobial activity of an SO2-releasing siderophore conjugate.
in Journal of inorganic biochemistry
Blagova E
(2023)
Thermostable homologues of the periplasmic siderophore-binding protein CeuE from Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius
in Acta Crystallographica Section D Structural Biology
Booth RL
(2020)
Artificial imine reductases: developments and future directions.
in RSC chemical biology
Coulson B
(2020)
Carbon Nitride as a Ligand: Selective Hydrogenation of Terminal Alkenes Using [(?5 -C5 Me5 )IrCl(g-C3 N4 -?2 N,N')]Cl.
in Chemistry (Weinheim an der Bergstrasse, Germany)
Coulson B
(2019)
Carbon nitride as a ligand: edge-site coordination of ReCl(CO)3-fragments to g-C3N4.
in Chemical communications (Cambridge, England)
Edmonds KA
(2020)
1H, 13C, 15N backbone resonance assignments of the apo and holo forms of the ABC transporter solute binding protein PiuA from Streptococcus pneumoniae.
in Biomolecular NMR assignments
Hussein SM
(2024)
Identification of a system for hydroxamate xenosiderophore-mediated iron transport in Burkholderia cenocepacia.
in Microbiology (Reading, England)
Large B
(2021)
Artificial metalloenzymes: The powerful alliance between protein scaffolds and organometallic catalysts
in Current Opinion in Green and Sustainable Chemistry
Miller A
(2024)
Catch-and-Release: The Assembly, Immobilization, and Recycling of Redox-Reversible Artificial Metalloenzymes
in ACS Catalysis
Miller LM
(2019)
Surface-Bound Antibiotic for the Detection of ß-Lactamases.
in ACS applied materials & interfaces
Raines D
(2018)
Redox-switchable siderophore anchor enables reversible artificial metalloenzyme assembly
in Nature Catalysis
Raines DJ
(2016)
Bacteria in an intense competition for iron: Key component of the Campylobacter jejuni iron uptake system scavenges enterobactin hydrolysis product.
in Proceedings of the National Academy of Sciences of the United States of America
Sanderson TJ
(2020)
A Salmochelin S4-Inspired Ciprofloxacin Trojan Horse Conjugate.
in ACS infectious diseases
Southwell J
(2023)
Augmentation of growth media for extreme iron-limitation in Escherichia coli
Southwell JW
(2021)
Experimental Methods for Evaluating the Bacterial Uptake of Trojan Horse Antibacterials.
in ChemMedChem
Southwell JW
(2023)
Siderophore-Linked Ruthenium Catalysts for Targeted Allyl Ester Prodrug Activation within Bacterial Cells.
in Chemistry (Weinheim an der Bergstrasse, Germany)
Wilde EJ
(2017)
Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM4- siderophore analogues of varied linker length.
in Scientific reports
Wilde EJ
(2019)
Mimicking salmochelin S1 and the interactions of its Fe(III) complex with periplasmic iron siderophore binding proteins CeuE and VctP.
in Journal of inorganic biochemistry
Zhang Y
(2020)
The Pneumococcal Iron Uptake Protein A (PiuA) Specifically Recognizes Tetradentate FeIIIbis- and Mono-Catechol Complexes.
in Journal of molecular biology
Description | Artificial metalloenzymes that consist of a synthetic catalyst that is linked via an anchor to a protein scaffold allow the bio-orthogonal reactivity of organometallic catalysts to be combined with the selectivity of enzymes. A particular strength of this approach lies in the synthesis of enantiomerically pure compounds. To complement the commonly used biotin-(streptavidin) affinity pair, we have identified ferric siderophore-binding protein pairs that allow organometallic catalysts to be anchored with suitably high affinities. Siderophores are ligands that are produced by bacteria for the uptake of essential iron and hence, ferric siderophore complexes are bound both strongly and enantioselectively by their cognate periplasmic binding proteins. By linking iridium-based transfer hydrogenation catalysts to selected siderophore-binding protein systems, the reduction of prochiral imines to chiral amines was achieved with encouraging levels of enantioselectivity. An exciting, but still unrealised advantage of the anchoring approach is its potential for controlled reversibility and thus component recycling. The iron(III)-binding siderophore-protein affinity pair that was developed with the help of this grant enables strong but redox-reversible catalyst anchoring, as we exemplified with our artificial transfer hydrogenase. By linking the siderophore azotochelin to an iridium-containing imine-reduction catalyst that produces racemic product in the absence of the binding protein CeuE, but a reproducible enantiomeric excess if protein bound, the assembly and reductively-triggered disassembly of the artificial enzyme was achieved. Whilst in the presence of iron(III) the azotochelin-based anchor binds CeuE with high affinity, the reduction of the coordinated iron(III) to iron(II) triggers its dissociation from the protein. Thus, the assembly of the artificial enzyme can be controlled via the iron oxidation state. We anticipate that this redox-switchable nature of ferric-azotochelin anchoring, if combined with a modular approach to enzyme design, could extend the range of applications in which artificial enzymes can be utilised. |
Exploitation Route | The developed novel synthetic procedures, characterisation data of novel compounds and catalytic reaction conditions have been published: D. J. Raines, O. V. Moroz, E. Blagova, J. P. Turkenburg, K. S. Wilson, A.-K. Duhme-Klair, PNAS, 2016, 113, 5850 and D. J. Raines, J. E. Clarke, E. V. Blagova, E. J. Dodson, K. S. Wilson, A.-K. Duhme-Klair, Nature Catalysis, 2018, 1, 680. Relevant data sets are open access and available to other researchers on PURE. |
Sectors | Chemicals,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
URL | https://rdcu.be/4RgH |
Description | It has been recognised that the redox-reversible design of our artificial systems has the potential to significantly extend the range of applications in which artificial metalloenzymes can be used. Hence, further research, supported via EP/T007338/1, is exploring the wider scope and applicability of the 'catch-and-release' approach. The techniques and skills developed during the work were passed on two other PDRAs, graduate and undergraduate students and opened up avenues for new collaborations and inspired several related projects, for example the development of assays for the profiling of susceptibility to antibiotics (EP/P02324X/1) and the immobilisation of transfer hydrogenation catalysts on heterogeneous supports that impose restricted steric environments. The former has led to an invention for which IP has been obtained (GB application No 2008603.9). In addition, we have produced an educational online resource aimed at undergraduate and graduate students: Siderophores, In: Reedijk, J. (Ed.), Elsevier Reference Module in Chemistry, Molecular Sciences and Chemical Engineering, Waltham, MA:Elsevier, 2015 doi: 10.1016/B978-0-12-409547-2.11040-6. |
First Year Of Impact | 2015 |
Sector | Chemicals,Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Impact Types | Societal,Economic |
Description | 21ENGBIO: In Cell Assembly of Artificial Imine Reductases for Whole-Cell Catalysis |
Amount | £100,515 (GBP) |
Funding ID | BB/W011131/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 02/2023 |
End | 02/2024 |
Description | Centre for Future Health |
Amount | £39,000 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 02/2018 |
End | 09/2018 |
Description | EPSRC DTG studentship |
Amount | £44,331 (GBP) |
Funding ID | EP/N509802/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2018 |
End | 09/2023 |
Description | EPSRC Healthcare Impact Partnerships |
Amount | £1,127,252 (GBP) |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2017 |
End | 08/2020 |
Description | Redox-reversible artificial metalloenzymes |
Amount | £722,386 (GBP) |
Funding ID | EP/T007338/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2020 |
End | 01/2023 |
Title | Dataset associated with publication: 'Interactions of periplasmic binding protein CeuE with Fe(III) n-LICAM4- siderophore analogues of varied linker length' |
Description | The dataset provides data associated with CD spectroscopic measurements, fluorescence titration curves and mass spectra. |
Type Of Material | Data analysis technique |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | The dataset allows other researchers to utilise the data, thereby facilitating further work in the area. |
Title | Dataset for publication: 'Redox-reversible siderophore anchor enables reversible metalloenzyme assembly' |
Description | Characterisation data of compounds 8-12 and S2, intrinsic fluorescence titration data, UV/vis data associated with reversibility studies, catalytic data associated with table 1 and SI Fig. 9b. |
Type Of Material | Database/Collection of data |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | The availability of the data associated with the publications allows other reserachers to use the information, hence facilitating further work in this and related areas. |
Title | Dataset relating to publication:'Bacteria in an intense competition for iron: key component of the C. jejuni iron uptake system scavenges enterobactin hydrolysis product' |
Description | The dataset contains NMR, IR and mass spectra and data files relating to the electronic absorbance, fluorescence and circular dichroism spectroscopic investigations. |
Type Of Material | Database/Collection of data |
Year Produced | 2015 |
Provided To Others? | Yes |
Impact | The data are available to other researches to enable and facilitate further work in the area. |
Description | ACS Meeting in Orlando, USA: Award Symposium in honor of Prof. Debbie Crans |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | About 50 academics and their associated groups came together to celebrate Prof Debbie Crans' contributions to the advancement of the inorganic chemistry scientific community. The symposium provided excellent networking opportunities and it sparked a wide range of discussions. |
Year(s) Of Engagement Activity | 2019 |
URL | https://natsci.source.colostate.edu/debbie-crans-receives-acs-award-for-distinguished-service-in-the... |
Description | Cardiff |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Postgraduate students |
Results and Impact | Seminar presetation at the University of Cardiff: 'Iron on the move: using siderophores to deliver antimicrobials and catalysts', 07/01/2019. |
Year(s) Of Engagement Activity | 2019 |
Description | International Conference on Coordination Chemistry, Brest, France, July 2016 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Oral presentation in the Bioinorganic Chemistry session of the conference |
Year(s) Of Engagement Activity | 2016 |
Description | King's College London |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Postgraduate students |
Results and Impact | Presented a seminar on iron scavengers and how to exploit them to the graduate at King's College London |
Year(s) Of Engagement Activity | 2018 |
Description | Norwich Research Park, Doctoral Training Partnership Summer Conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | 180 Participants, including NRP Doctoral Training Partnership students, students affiliated to the programme and MRC Doctoral Antimicrobial Resistance Training (DART) iCASE students attended. The topic of the event was Frontier Bioscience. A number of interesting questions resulted in a lively discussion. |
Year(s) Of Engagement Activity | 2019 |
URL | https://biodtp.norwichresearchpark.ac.uk/news/successful-nrpdtp-summer-conference-2019/ |
Description | Research seminar, University of Sussex |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | Postgraduate students, PDRAs and members of staff attended the research seminar. In addition, a number of 1:1 meeting with researchers resulted in knowledge exchange and raised interesting questions for further investigations. Future funding opportunities were discussed. |
Year(s) Of Engagement Activity | 2019 |
Description | Scottish Dalton Meeting (Heriott-Watt University, Edinburgh) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | The Scottish Dalton regional meeting 2019 was organised by the RSC and attended by approx. 150 inorganic chemistry researchers, including academics, PDRAs and graduate students. It resulted in a lively scientifc discussion and exchange of experiences and research ideas, which was supported by a poster session. |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.rsc.org/events/detail/36259/scottish-dalton-meeting |
Description | UEA |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Postgraduate students |
Results and Impact | Seminar presentation at the University of East Anglia: 'Iron on the move: using siderophores to deliver antimicrobials and catalysts', 21/11/2018. |
Year(s) Of Engagement Activity | 2018 |
Description | University of Leeds |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Postgraduate students |
Results and Impact | Seminar presentation on 'Iron on the move: using siderophores to deliver antimicrobials and catalysts' at the School of Chemistry, University of Leeds |
Year(s) Of Engagement Activity | 2018 |
Description | University of Sheffield, Department of Chemistry, Christmas Lecture |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | The Molecular Scale Engineering CDT, which is split between the Universities of Leeds and Sheffield, invites a speaker to give a Christmas lecture every year. In 2016, the event was hosted at the University of Sheffield. Since the CDT consists of Graduate Students from across disciplines, including chemistry and physics, the Christmas lecture was aimed at a broad audience. The title of the lecture was: 'Bacterial iron scavengers and how to exploit them'. |
Year(s) Of Engagement Activity | 2016 |
Description | York Festival of Ideas: An artistic exploration of antibiotic resistance |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | The aim of the workshop, which was held as part of the York Festival of Ideas, was to raise awareness of the problem of antimicrobial resistance and the need for new treatments. Held in collaboration with bioartist Anna Dumitriu, it included an exhibition of artwork that was created in the laboratory by using bacteria. Visitors had the opportunity to discuss their experience with antibiotics and antimicrobial resistance with microbiologists, medicinal chemists and the artist. In addition, visitors were encouraged to participate in both science experiments and the production of artwork. The latter was then integrated into the exhibition. Over two days, a variety of visitors engaged with the activity, which brought audiences interested in art and/or science together. The unusual mix of expertise sparked very interesting discussions. |
Year(s) Of Engagement Activity | 2016 |