The need for health improvement on the African continent continues to be a pressing issue, and START’s emphasis will be on diseases such as HIV-AIDS, malaria, tuberculosis, and African horse sickness that are devastating to human and animal populations. The structural biology strand of START research will support scientists in finding and developing cures by researching and understanding the fundamental molecular structure of certain diseases. Prof. Trevor Sewell from the University of Cape Town explains:
“START will allow us to understand drug targets and cure African diseases. We will establish a collaborating network of seven South African institutions (the Universities of Pretoria, Witwatersrand, North West, Free State, Stellenbosch, Cape Town and the National Institute for Communicable Diseases) that will enable young researchers to boost medical and veterinary research”.
Prof. Trevor Sewell, University of Cape Town
A START project at University of Cape Town led by co-investigator Prof Edward D Sturrock
ACE in complex with the clinically used antihypertensive drug, lisinopril (black sticks; PDB ID: 1O86)
Structural biology of angiotensin converting enzyme and related metalloproteases
Enzymes play important roles in a variety of biological processes in the human body. Angiotensin converting enzyme (ACE) for example, is a metalloprotease which regulates blood pressure and is also involved in scar tissue development (fibrosis). Conditions such as diabetes and tuberculosis can lead to excessive scar tissue formation, which ultimately stops proper organ function. Currently, there is no specific treatment for fibrosis and affected individuals have an average survival period of 2-4 years. Hypertension, on the other hand, is a major risk factor for cardiovascular disease and stroke, which accounted for 15.2 million global deaths in 2016. Our research group, led by Prof Edward Sturrock, is based in the Department of Integrative Biomedical Sciences at the University of Cape Town and has a long-standing interest in ACE and related zinc metalloproteases.
Although ACE inhibitors reduce fibrosis and are widely used for treating hypertension, certain patients experience the life-threatening side-effect of severe swelling below the skin surface of the throat and tongue. With the resources provided by START, we aim to design compounds devoid of this side-effect. Detailed structures of ACE will be solved using X-ray crystallography and cryo-electron microscopy to improve our understanding of how ACE functions and enable the design of antifibrotic and antihypertensive drugs.
START Collaborators – research projects
For information on projects please click on the names below
Visualising the structure of an intact helical filament at close-to-atomic resolution for the first time
“We are seeing critical scientific discoveries and the emergence of a new generation of experts that have resulted directly from our training programmes in advanced methods and the use of synchrotron facilities and tools.” Dr Gwyndaf Evans, START Principal Life Sciences Principal Investigator and Principal beamline scientist on Diamond’s VMXm beamline.
A seminal work of Dr Jeremy Woodward, Dr Andani Mulelu and Angela M.Kirykowicz from the University of Cape Town (UCT), South Africa, has provided novel and exciting insights into the structure and inner workings of nitrilase enzymes with the potential to address key health, food security and environmental challenges within Africa and beyond.
The results were published on the 17 July 2019 in Nature Communications Biology 2:260 (2019)4 and made possible through the UK’s national synchrotron light source, Diamond Light Source, which has integrated facilities for life sciences users as a ‘one stop shop’ for structural biology.
The first high resolution visualisation of a Cryo-EM6 protein structure in Africa
Nitrilases are a class of plant enzymes that play an important role in the synthesis of a broad range of chemicals. Although have specificity for a small range of substrates they have a large potential to create products of biotechnological significance.
Building on more than a decade of structural biology research, and exploiting knowledge sharing and synchrotron access opportunities through the Global Challenges Research Fund’s GCRF START Programme, Dr Woodward, Dr Mulelu and Angela Kirykowicz were able to visualise the structure of an intact helical filament at close-to-atomic resolution for the first time – the first high resolution visualisation of a Cryo-EM6 protein structure ever to be produced in Africa (Fig 1).
The scientists achieved this at the UK’s national Electron Bio-Imaging Centre (eBIC), using Cryo-Electron Microscopy, on the Titan Krios III (Beamline M06) – one of Diamond’s ‘super microscopes’ – to observe how the maximum size of a bound substrate is limited by a loop which shifts with helical twist after mutating a single amino acid.
Observing the ‘loop’, ‘lock’ and ‘lid’: Dr Woodward describes their observations in terms of a ‘loop’, ‘lock’ and ‘lid’: the size of the binding pocket seems to be limited by a ‘lid’, which prevents long substrates from being converted. This lid is formed by a ‘loop’ which is held in position by electrostatic force. A single amino acid is responsible for maintaining this interaction – this is referred to as the ‘lock’. Other amino acids within the binding pocket interact with various chemical groups of the substrate and either allow it to bind or prevent it from binding. Two regions in particular are important for this effect. The three amino acids are: “the lock”, which defines the overall size of the substrate that can bind (either by tightening or loosening the lock by altering its chemical properties); and two others within the binding pocket, which interact with the substrate directly.
The insights gained allowed the team to semi-rationally design a new mutant nitrilase enzyme that produces biotechnologically interesting products, whether pharmaceuticals, fine chemicals or even food.
“We did this by identifying ‘hot spot’ amino acids for directed evolution and selecting them by coupling the survival of bacteria to the successful conversion of a library of substrates,” explains Dr Woodward, who had identified the most important of these residues ten years ago.
“Previously, at low low-resolution, we had seen large-scale changes,” Dr Mulelu adds “but we needed to answer the question: “How did this work? How did these translate into differences that could account for the ability of these enzymes to distinguish between substrates that differ by only one carbon atom? The Titan Krios III (beamline M06) housed at eBIC enabled us to answer our questions and see the enzyme at atomic scale resolution and to achieve these wonderful results.”
These results pave the way for further exciting research opportunities. Going forward, Dr Woodward’s ultimate aim is to produce a ‘catalogue’ of ‘designer’ nitrilases’ in a quest to find solutions through biotechnology which could lead to sustainable transformations in the lives and livelihoods of populations across Africa.
“My vision is to create a ‘catalogue’ of ‘designer nitrilases’ for any substrate by making appropriate changes to the helical twist as well as the binding pocket,” Dr Woodward says. “To achieve this, we would like to visualise a collection of key nitrilases with a range of different helical states (and substrate specificities). We are currently using computer modelling to predict binding energies and correlate these with known substrate specificities of various binding pocket mutants we have.”
Meeting the global challenges
The results achieved by the UCT team could play an important part in providing sustainable solutions in the future to meet the UN’s Sustainable Development Goals, from novel drug design and manufacturing to tackle communicable and non-communicable diseases, to pioneering ‘green’ biotechnology options for agricultural food security, industrial and mining waste treatment using enzyme-catalysed products.
Medical drug discovery and manufacturing
Dr Woodward is convinced that investments in new drug manufacturing methods may have a bigger impact on health on the African continent than the development of entirely new drugs,
”Africa has the worst burden of life-threatening communicable diseases in the world,” explains Dr Woodward. “According to the UN, 1.6 million people died from a combination of preventable and treatable diseases in 2015 – malaria, tuberculosis and HIV-related disease. A big problem in Africa, however, is access to medicines and not necessarily the development of new medicines. Part of the problem is that only 2% of the medicines used in Africa are manufactured in Africa, which has implications for the cost and accessibility of medicines.”
This is where nitrilase enzymes could provide solutions. Nitrilase enzymes are attractive biocatalysts for the synthesis of amides and carboxylic acids for use in the manufacture of drugs for major life-threatening communicable diseases, such as malaria, tuberculosis and HIV-related disease.
Drug discovery and testing for these diseases would also benefit from the economies of scale of an ‘off the shelf’ catalogue of nitrilases manufactured in Africa, making drugs for these diseases much more accessible and affordable.
“The ability to manipulate how nitrilases work,” Dr Mulelu points out, “could enable more cost-effective manufacturing of drugs for such diseases, leading to cheaper and more accessible medicines and this is welcome news for us in Africa and in other developing countries.
In addition, manufacturing locally has a variety of advantages including lowering costs of transport and improving local economic development. Nitrilases can offer ‘green’ alternatives because the reaction occurs at (close to) room temperature, neutral pH and atmospheric pressures so require less energy to produce. They are also very specific and so produce less waste.”
‘Green biotechnologies’ for agriculture and waste treatment
Looking beyond pharmaceuticals, nitrilases have a host of potential ‘green technology’ solutions to offer for cleaning up environmental pollution. One example is the breaking down of cyanide, which could revolutionise the way we clean hazardous mining dumps, reduce water pollution and improvements in the treatment of industrial waste and green manufacturing using enzyme-catalysed products.
“The nitrilase 4 mutant we have produced (described in the latest paper) can break down cyanide, forming products that can be further broken down by bacteria,” Dr Woodward explains. “This also applies to farming and food security. These compounds release cyanide and animals, especially ruminants, can suffer cyanide poisoning as a result but nitrilases with specificity towards cyanide could be used in a genetically modified strain of sorghum, which could break down the cyanide and make it safer.”
Developing a new generation of research leaders
A significant focus of the START programme focuses on capacity building and investing in the development of existing and future science talent across the continent, as well as funding Post-Doctoral Research Assistants and Fellows, and resourcing laboratories. For Dr Andani Mulelu, the impact of being a ‘Synchrotron Techniques for African Research and Technology (START) Postdoctoral Research Fellow’ has been significant, particularly in achieving results,
“During my honours year I became fascinated by the structure of helical nitrilases, especially those that detoxified cyanide, and I joined the group of Professor Trevor Sewell for my Masters and later my PhD. Working with Dr Jeremy Woodward and GCRF START enabled me to finally to realise my dream of visualising a nitrilase at atomic resolution and to solve the mystery of substrate selectivity in these enzymes.”
Dr Mulelu was subsequently offered a job as a research scientist at the H3D Drug Discovery and Development Centre (UCT) working on Malaria and Tuberculosis target-based drug discovery programmes, a move he attributes to the experience he gained as a START-funded post-doc.
Ms Kirykowicz was a Masters student at the time the team achieved the successful results described in the Nature paper. Interested in applying the directed evolution techniques from her Honours year, Ms Kirykowicz’s role in the team involved working on nitrilase specificity, which gave her the skills she needed her future studies,
“The START-funded project gave me a good understanding of the methods needed to produce a well-sampled mutant library. I liked structural biology and ended up completing my Master’s on solving Mycobacterial protein complexes with Dr. Woodward. I am currently doing a PhD at the University of Cambridge in the UK working on solving the cryo-EM structure of a protein toxin transporter.”
