Appeal Against the Decision Not to Fund the Proposal Nitrile Metabolizing Enzymes

Private Bag Rondebosch 7701 South Africa Telephone: +27 (0)21-650-2817 Facsimile: +27 (0)21-689-1528 Email: [email protected]

Appeal against the decision not to fund the proposal Nitrile metabolizing enzymes

Our previous NRF grants in this area have enabled us to establish an incredibly successful programme. The study of nitrile metabolizing enzymes has become our main line of research and without this funding it cannot continue. It was the recommendation of the rating committee that reviewed my successful rating application that I concentrate my effort in this area. At present the research of four PhD students: Ms Serah Kimani, Mr Jeremy Woodward, Mr Ndoria Thuku and Ms Joni Frederick, and two Masters Students: Mr Johann Eicher and Mr Kyle Dent. Our twelve publications in this area are being widely cited and we have made some important discoveries, notably: the unusual spiral nature of the nitrilases, the reversible pH dependent helix formation, the residues at the subunit interfaces, the hexameric arrangement of the amidases, the involvement of a second glutamate in the catalytic activity of nitrilases and amidases, the presence of a second sulfinic acid in the Co containing nitrile hydratases etc. - most notably our discoveries on the mechanism which may open the way for new drugs to combat tuberculosis and possibly malaria.

My prominence in the field is attested by the fact that I have been invited to give a plenary lecture on these enzymes at a Gordon conference - widely regarded as being prestigious international conferences.

The enzymes are of industrial importance and there is a real possibility that they will be utilized industrially in South Africa as a result of our research. The structural work we have done on these enzymes has established the field of Structural Biology locally and has put South Africa on the map in that discipline.

Many students have graduated with MSc's as a result of their studies in this field (Mr Mark Berman, Mr Ndoria Thuku, Mr Jeremy Woodward, Ms Serah Kimani, Ms Margot Scheffer, Mr Sam Kwofie). Miss Serah Kimani in particular - whose further studies would have been funded out of this grant has been awarded this year's S2A3 bronze medal as the best MSc in South Africa. Two of our papers in this area have been chosen for cover pictures on the journals in which they have been published - giving substantial extra international publicity to our work.

Furthermore the NRF has recently granted a major piece of equipment necessary for further work in this area i.e. the FEI F20 electron microscope - which was motivated on the basis of this work and to further this work. It will not be possible to exploit the instrument without funding for the projects.

Our published papers in this area are as follows:

Jandhyala, D., Berman, M., Meyers, P.R., Sewell, B.T., Willson, R.T.,and Benedik, M.J. (2003). CynD, the Cyanide Dihydratase from Bacillus pumilus: gene cloning and structural studies. Appl. Environ. Microbiol. 69, 4794-4805.

Sewell, B.T., Berman, M., Meyers, P.R.,Jandhyala, D., and Benedik, M.J. (2003). The Cyanide Degrading Nitrilase from Pseudomonas stutzeri AK61 Is a Two-Fold Symmetric, 14-Subunit Spiral, Structure, 11, 1413-1422.

Tsekoa, T.L., Sayed, M.F., Cameron, R.A., Sewell, B.T. and Cowan D.A. (2004) Purification, crystallization and preliminary X-ray diffraction analysis of thermostable nitrile hydratase. South African Journal of Science, 100, 488-491. Jandhyala,DM, Willson,RC, Sewell,BT and Benedik,MJ.(2005). Analysis of Three Microbial Cyanide Degrading Enzymes. Applied Microbiology and Biotechnology. 68, 327-335 Sewell, BT, Thuku RN, Zhang X, Benedik M. (2005) The oligomeric structure of nitrilases: the effect of mutating interfacial residues on activity. Annals of the New York Academy of Sciences, 1056, 153– 159

Tastan Bishop AO, Sewell T (2006) A new approach to possible substrate binding mechanisms for nitrile hydratase. Biochem Biophys Res Commun. 343,319-25.

Agarkar VB , Kimani, SW, Cowan, DA, Sayed, MF-R., Sewell, BT (2006) The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing, Acta Cryst. F62 , 1174-1178

Makhongela HS, Glowacka, A., Agarkar VB, Sewell, BT, Weber, B, Cameron RA., Cowan DA, and Burton SG (2007) Molecular characterization and immobilization of D-specific amidase from Geobacillus pallidus. Applied Microbiology and Biotechnology, 75, 801-11

Thuku, RN, Weber, BW, Varsani, A and Sewell, BT (2007) Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form. FEBS Journal, 274, 2099-2018

Kimani, SW, Agarkar,VB Cowan, DA, Sayed, MF-R and Sewell, BT (2007) Structure of an aliphatic amidase from Geobacillus pallidus RAPc8 Acta Cryst. D63, 1048–1058 Woodward, J.D.; Weber, B.W.; Scheffer, M.P.; Benedik, M.J.; Hoenger, A.; Sewell, B.T. (2008) Helical structure of unidirectionally shadowed metal replicas of cyanide hydratase from Gloeocercospora sorghi. J Struct Biol, In Press: PMID: 17997328.

Seven more papers are in preparation, three are at an advanced stage. There are no alternate funds for this research. I sincerely hope that the decision not to fund this grant will be reconsidered - otherwise no further work can be done in this area by us.

Yours sincerely,

Trevor Sewell Director of the Electron Microscope Unit