Collaborations

Prof. Felix Carvalho and Dr. Juliana Garcia
Faculdade de Farmácia, Universidade do Porto, Portugal

Prof. Armando Silvestre e Dr. Andreia Sousa
CICECO- Universidade de Aveiro, Portugal

Prof. Marcel Swart
Girona University, Spain
 
Prof. Kristala Prather
Massachusetts Institute of Technology, US

The research in the Rational protein engineering group is focused in the design of enzymes with: 1) increased activity; 2) improved or inverted selectivity; 3) enhanced structural stability. Computational biochemistry methods, which include molecular docking, molecular dynamics and Quantum mechanics/Molecular mechanics calculations are employed using our own protocols in the study of enzyme binding, catalysis and dynamics as well as in the design of new variant enzymes with the desired properties. The rationally designed enzymes are obtained at fraction of the time and cost when compared with traditional directed evolution protocols like: error prone polymerase chain reaction (epPCR); DNA shuffling (DS); saturation mutagenesis (SM), which are normally associated with the screening of ~105 clones.

We are pursuing three lines of research:
1)    Development of new endonucleases with high activity and low off-target effects for gene editing
The repair of the disease causing mutations by the use of gene editing technology holds enormous potential in the near future. Several endonucleases have been tested for gene editing with the most promising so far being the Cas9 protein. However, despite its enormous potential, this enzyme is error prone and has a tendency to cleave dsDNA at off target sites that are not fully complementary to the guide RNA This can result in an unexpected mutations or rearrangements of the genome which might lead to oncogenesis. We are studying endonuclease models and rationally designing them to improve specificity while maintaining high catalytic efficiency in the complex task of genome editing.

2)    Development of antidotes for alpha-amanitin poisoning
The deadly amatoxin alpha-amanitin, which is present is some mushrooms, is known to bind to RNA polymerase II preventing transcription. We have been involved in a collaborative project with the toxicology group from the Faculty of Pharmacy (Porto University) to develop an effective antidote of  alpha-amanitin  poisoning, which can bind the RNAP amanitin binding site without compromising DNA transcription.

3)    Development of new enzymes for the synthesis of biobased polyesters
Plastics (fossil fuel derived polymers) are indispensable for our current lifestyle. However, plastic pollution is causing irreparable damage to the environment. In 2015 the amount of plastic waste was estimated at 6300 million metric tons (Mt), with 79% present in landfills  or the natural environment. It current trends continue, around 12,000 Mt will be present in landfills or the natural environment by 2050 (Geyer et al, Science Advances, 2017).  It is thus urgent to find more sustainable alternatives. We are developing enzymes for the biosynthesis of biodegradable polyesters from bio-based sources.

Selected publications:

1.    Dourado D. F. A. R., Swart M., Carvalho A. T. P. Why the flavin dinucleotide cofactor needs to be covalently linked to Complex II of the electron transport chain for conversion of FADH2 to FAD. Chem. – Eur J. 2017.

2.    Carvalho, A. T. P., Dourado, D. F. A. R., Skvortsov, T., de Abreu M., Ferguson, L. J., Quinn, D. J., Moody, T. S. Huang, M. Spatial requirement for PAMO for transformation of non-native linear substrates. Phys. Chem. Chem. Phys. 2018, Advance Article.

3.    Carvalho, A. T. P., Dourado, D. F. A. R., Skvortsov, T., de Abreu M., Ferguson, L. J., Quinn, D. J., Moody, T. S. Huang, M. Catalytic mechanism of phenylacetone monooxygenases for non-native linear substrates. Phys. Chem. Chem. Phys. 2017, 19, 26851–26861. IF=5.3

4.    Dourado, D. F. A. R., Pohle, S., Carvalho, A. T. P., Dheeman, D. S., Caswell, J. M, Skvortsov, T., Miskelly, I., Brown, R. T., Quinn, D. J., Allen, C. C. R., Kulakov, L., Huang, M., Moody, T. S. Rational design of a (S)-selective-transaminase for asymmetric synthesis of (1S)-1-(1,1’-biphenyl-2-yl)ethanamine. ACS Catalysis 6, 7749-7759 (2016). IF=9.3

5.    Garcia J., Costa V.M., Carvalho A. T. P., Dourado D., Silvestre R., Dinis R., Arbo M., Baltazar T.,Baptista P., Bastos L.M., Carvalho F. A breakthrough on Amanita phalloides poisoning: an effective antidotal effect by polymyxin B. Archives of Toxicology, 11-19 (2015). IF=5.98

Patents

WO2016185403 A1- Use of polymyxin as an antidote for intoxications by amatoxins. Inventors: Félix Carvalho, Juliana Garcia, Maria Lourdes Bastos, Vera Marisa Freitas Costa, Alexandra Carvalho, Ricardo Silvestre, Alberto Ramos Duarte Jose, Jorge Dinis Oliveira Ricardo. 24 Nov 2016.

CV

2017-present: Assistant Investigator at Center for Neuroscience and Cell Biology (CNC)/Coimbra

2015-2016: Researcher at Almac Sciences/Queen’s University Belfast, Northern Ireland   

2013-2015: Senior Researcher at the Kamerlin lab, Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Sweden  

2012–2013: Marie Curie Fellow (Beatriu de Pinós) at the Institute of Computational Chemistry, Girona University

2009-2012: Posdoctoral Fellow at the Computational Chemistry group, Chemistry department, Porto University

2004-2008: PhD in Computational Chemistry, Porto University  

1999-2003: Degree in Biochemistry, Chemistry Department and Abel Salazar Biomedical Institute, Porto University