Instruct-ERIC and START: Health & Bio Science have opened doors to supportive research platforms, networks, and experts, not only for my current project but for my skills training and career.
Lenye Dlamini, University of Cape Town, South Africa
The problem
The growing evidence of the negative effects of human activities on environments and communities in Africa1 and globally, has necessitated studies into sustainable, efficient, and environmentally friendly alternatives to conventional chemical processes and methods to remediate damaged ecosystems. A common pollutant is cyanide that is used in gold extraction and electroplating. It also arises in landfill sites because of natural processes. Cyanide can be converted to environmentally safe formic acid by the action of bacterial and plant derived enzymes called nitrilases.
An issue is that nitrilases cannot withstand the conditions they encounter in environmental or industrial use. They need to have their stability improved by making appropriate alterations to their structure without changing their activity. In addition, nitrilases have well-known uses as industrial biocatalysts in the synthesis of both commodity and fine chemicals. There is also a need to increase their stability in this context.
The challenge
Stable, industrially useful nitrilases are generally discovered by searching in the environment and then tailoring the discovered enzyme by a process called ‘directed evolution’. Trevor Sewell’s laboratory at the University of Cape Town has been doing this for some 20 years, but the question that arises is: can the stability-inducing mutations be correctly predicted by computer if the enzyme structure is known?
In the case of nitrilases, in which the active enzyme form is a helix made up of multiple repeats of the same protein, this prediction is challenging as the stability is dictated by both interactions within the repeating molecules and between them. The parameter that describes the stability is called the ‘Gibbs free energy’. This can be computed if the structure is known and measured by a variety of chemical and physical techniques.
The structure of nitrilase fibres is best determined by cryo-electron microscopy (cryo-EM), a technique for which only rudimentary equipment exists in Africa. Research initiated at UCT led to the computation of maps of a bacterial cyanide degrading enzyme and a thermostable variant of this enzyme called Q86R by, among others, Dr Andani Mulelu.
Once the structure is known, the Gibbs free energy can be calculated, and mutations can be made in silico. The process can be repeated iteratively until supposedly more stable protein variants are found. The protein can then be made, and the Gibbs free energy can be measured by a variety of different methods, the most convenient of which is label free nano differential scanning fluorimetry, another technique that is not available in Africa.
The solution
Lenye Dlamini is a PhD candidate at UCT working on a project2 to address the problem by engineering a cyanide-degrading nitrilase with enhanced stability. Nitrilases have intrinsically robust, spiral structures that suggest that substantial improvements in stability are possible. High resolution structures will enable Lenye to understand the chemical and structural basis for enzyme function and – very important in this case – the basis for oligomerisation, which is linked to stability and therefore tolerance to thermal stress and high pH.
To date, Lenye has interpreted the structure of cyanide dihydratase from Bacillus pumilus C1 and has deposited the map and model of the Q86R variant in the Electron Microscopy Database (EMDB). Several mutations that may contribute to enhanced thermostability of this enzyme have been computationally identified. Lenye is investigating their effects experimentally using cryo-EM and biophysics techniques such as nano-differential scanning fluorimetry, allowing comparisons and correlations between computational and experimental data to be made.
Substantial funding has been secured to support Lenye and her research. Funding sources include: GCRF START grant, Instruct-ERIC, The LÓreal For Women in Science (FWIS) and Organisation for Women in Science for the Developing World (OWSD), the International Union of Crystallography, the National Research Foundation of South Africa, and most recently, START: Health & Bio Science.
Impact
Although the research is still in its early stages, several scientific and capacity building milestones have been achieved. The Instruct-ERIC international access call https://instruct-eric.org/news/instruct-eric-announces-2nd-international-call-awardees/ has enabled Lenye to conduct key aspects of her research at the Membrane Protein Laboratory at the Research Complex on the UK’s Harwell Science and Innovation campus, where she has used the state-of-the-art cryo-EM Glacios instrument for her experiments. Having direct access to the latest cryo-EM and biophysics technology in the UK has allowed Lenye to explore parts of the nitrilase project that would have been impossible in her laboratory in South Africa. With expert knowledge and equipment immediately at hand, Lenye’s PhD research has accelerated.
Lenye’s research directly addresses several UN’s Sustainable Development Goals, including responsible and sustainable consumption and production (SDGs 9 & 12) and South Africa’s renewed commitment to a proactive and sustainable response to global environmental and climate change (SDG 13), and the reduction of human impact on our environment (SDGs 6, 14 & 15).
Capacity building
Lenye was selected for the European Molecular Biology Organisation (EMBO) course on image processing for electron microscopy, the CCP4 Crystallographic School in South Africa, Data Collection to Structure Refinement and Beyond, Becoming an ‘independent’ single particle data collector, and Centre for High Performance Computing South Africa (CHPC) summer schools giving her unprecedented opportunities to meet and be guided by the internationally renowned scientists and the authors of the software that she is using. These opportunities contribute towards the UN’s Sustainable Development Goals for quality education (SDG 4) and gender equality (SDGs 5 & 10).
“Instruct-ERIC and START: Health & Bio Science have opened doors to supportive research platforms, networks, and experts, not only for my current project but for my skills training and career. LÓreal For Women in Science and Organisation for Women in Science, in conjunction with UNESCO, have also been pivotal. Both have gone beyond providing funding, giving me access to resources that are shaping me into a well-rounded scientist, and exposing me to communities of formidable and inspiring scientists that I would otherwise not have been part of.”
Lenye Dlamini, University of Cape Town, South Africa
Footnotes
1 See https://www.unep.org/regions/africa/our-work-africa accessed 14.3.2023