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  BBSRC NLD Doctoral Training Partnership: Cracking the metalation code – maturation of a heterologously expressed microbial metalloenzyme


   Department of Biosciences

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  Dr P.T. Chivers, Prof F Sargent  No more applications being accepted  Competition Funded PhD Project (UK Students Only)

About the Project

How does an enzyme get its correct metal? This answer is not simple. Nearly half of enzyme reactions require a metal cofactor but there is no universal code for that ensures the correct metal is always bound. For example, the heterologous expression in Escherichia coli of a metalloenzyme from another organism does not predictably lead to correct metalation. A detailed understanding of the cellular conditions that define metalation will have impacts on both fundamental and applied research. We study the molecular mechanisms used by enzymes and proteins to bind the correct metal using a combination of protein structure-function analysis (eg enzyme assays with purified native and mutant proteins) and molecular microbiology to improve metalation under heterologous expression conditions (common in industrial biotechnology).

This project uses nickel superoxide dismutase (NiSOD) as a model system for heterologous expression. NiSOD is encoded by two abundant microbes in the biosphere, Streptomyces and some cyanobacteria (estimated at 1025 cells in marine environments). The assembly of functional NiSOD involves an interesting proteolytic mechanism that requires formation of a complex between the expressed enzyme (SodN), a nickel-small molecule complex (Ni•L), and subsequent cleavage by a peptidase (SodX).1 However, when expressed in E. coli, SodN processing is poor compared and little active enzyme is obtained compared to the original organism (eg cyanobacteria). The project will focus on determining how exactly this reaction occurs correctly, both with purified components in vitro and in bacterial cells, to provide insight in the metalation code.

Training

The project will provide in depth training in protein biochemistry/enzymology and molecular microbiology. Opportunities for skills development range from specific practical components (protein purification and enzymatic assays in the tube and the cell) to more general components (quantitative data analysis, hypothesis testing and experimental design), and collaboration with other groups as the project progresses. The project also links to the recently funded BBSRC ELEMENTAL Engineering Biology Hub, which will enable interactions with like-minded groups around the UK (at regular Hub meetings, for example).

Project trajectory and outcomes

There is flexibility within the project objectives to emphasise more detailed structure-studies or more work with microbial systems, or a mixture, depending on the interests of the successful applicant. Post-PhD career goals could also shape the project direction and skills emphasis (for example, academia, industrial biotechnology R&D). 

1. Basak, P, Cabelli, DE, Chivers, PT, Farquhar, ER, and Maroney, MJ. In vitro maturation of NiSOD reveals a role for cytoplasmic histidine in processing and metalation. Metallomics (2023) doi.org/10.1093/mtomcs/mfad054

HOW TO APPLY:

Applications should be made by emailing [Email Address Removed] with:

  • a CV (including contact details of at least two academic (or other relevant) referees);
  • a covering letter – clearly stating your first-choice project, and optionally 2nd ranked project, as well as including whatever additional information you feel is pertinent to your application; you may wish to indicate, for example, why you are particularly interested in the selected project(s) and at the selected University;
  • copies of your relevant undergraduate degree transcripts and certificates;
  • a copy of your passport (photo page).

A GUIDE TO THE FORMAT REQUIRED FOR THE APPLICATION DOCUMENTS IS AVAILABLE AT https://www.nld-dtp.org.uk/how-apply. Applications not meeting these criteria may be rejected.

In addition to the above items, please email a completed copy of the Additional Details Form (as a Word document) to [Email Address Removed]. A blank copy of this form can be found at: https://www.nld-dtp.org.uk/how-apply.

Informal enquiries may be made to [Email Address Removed] 

The deadline for all applications is 12noon on Monday 22nd July 2024.

Part-Time Study Options

All NLD DTP PhDs are available as part time or full time, with part time being a minimum of 50% of full time. Please discuss potential part time arrangements with the primary supervisor before applying to the programme.

Project CASE Status

This project is not a CASE project. While individual applicant quality is our overriding criterion for selection, the NLD DTP has a commitment to fund 8 CASE projects per year - as such, CASE projects may be favoured in shortlisting applicants when candidates are otherwise deemed to be equal or a consensus on student quality cannot be reached.

Biological Sciences (4) Chemistry (6)

Funding Notes

BBSRC NLD DTP programme – starting October 2024. UKRI provide the following funding for 4 years: • Stipend (2024/25 UKRI rate £19,237) • Tuition Fees at UK fee rate (2024/25 rate £4,786) • Research support and training grant (RTSG) Note - UKRI funding only covers UK (Home) fees.


References

1. In vitro maturation of NiSOD reveals a role for cytoplasmic histidine in processing and metalation. (2023) Metallomics in press, doi.org/10.1093/mtomcs/mfad054
2. Bacterial sensors define intracellular free energies for correct enzyme metalation. Nat Chem Biol 2019 15, 241-249. doi.org/10.1038/s41589-018-0211-4
3. Elucidation of the biosynthesis of the methane catalyst coenzyme F430. Nature 543, 78-82 (2017)
4. Activation of a [NiFe]-hydrogenase-4 isoenzyme by maturation proteases. (2020) Microbiology 166, 854-860. doi.org/10.1099/mic.0.000963
5. Identification of a stable complex between a [NiFe]-hydrogenase catalytic subunit and its maturation protease (2017) FEBS Lett 591, 338-347. doi.org/ 10.1002/1873-3468.12540
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