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Kinetic characterization and effect of mutations in the oligomeric interface of the Nucleoside Diphosphate Kinase from Leishmania major.

Grant number: 10/03761-4
Support Opportunities:Scholarships in Brazil - Master
Effective date (Start): March 01, 2011
Effective date (End): March 31, 2012
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal Investigator:Arthur Henrique Cavalcante de Oliveira
Grantee:Plínio Salmazo Vieira
Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil


The nucleoside diphosphate kinase (NDK) is an enzyme responsible for the production of nucleoside triphosphates and is involved in various cellular processes. The NDK is secreted into the extracellular environment and is considered a pathogenic factor in intracellular pathogens such as Mycobacterium tuberculosis, Vibrio cholerae and Leishmania amazonensis. This project aims to characterize the kinetic activity of recombinant enzyme nucleoside diphosphate kinase (NDK) from L. Major by reverse-phase chromatography and evaluate the effect of mutations on the oligomeric interface, relating the changes in the structure of the catalytic activity of the enzyme and the functional activity in interacting with macrophages. The specific objectives are: 1) site-directed mutagenesis of recombinant NDK (R17A, E28a, P97S, P100G, P100S-delta5C-delta5C-terminal and terminal) and analysis of the relevance these aminoacids in the oligomeric state and the consequence of the quaternary changes in the enzyme activity, 2) purification of mutant enzymes in resin of Ni2+ and obtainment of the kinetic parameters using a coupling enzyme system pyruvate kinase / lactate dehydrogenase, 3) Establishment of the analysis of the reaction by reverse-phase chromatography to obtain the kinetic parameters of nucleotide acceptors phosphate; 4) Characterization of secondary, tertiary and quaternary structures, by spectroscopic studies of circular dichroism, fluorescence and gel filtration, 5) Analysis of cytotoxicity by eATP and invasion of macrophages in the presence of NDKrec and mutants to evaluate the effect mutations in these two functional activities. This enzyme has been purified and their kinetic characterization was partially done by the proponent student of this project, during his undergraduate work. The kinetic parameters of phosphate donors will be made with a coupled system of enzymes (pyruvate kinase - lactate dehydrogenase) and chromatography reverse phase using HPLC with the reaction products will be established to obtain the acceptors parameters. The first mutant, K30A, has shown a decreased catalytic activity and is being initiated the quaternary structure characterization of the protein. A R17A mutation is suggested because the arginine also makes hydrogen bond with the neighboring monomer (Janin et al., 2000) as the lysine at position 30. A mutant E28A was described as important for the dimer interface and will be made to the L. major NDK. The Kpn loop is essential for maintenance of protein structure (Webb et al., 1995) and P97S and P100S mutations has been described by changing the position of the Kpn loop, destabilizing the trimer and directly interfering in oligomerization (Lascu et al. 1992). Furthermore, to evaluate the involvement of the C-terminal loop in the oligomerization, this will be reduced by 5 amino acids, including in the mutant P100G. The quaternary structure will be analyzed by gel filtration to confirm the changes in oligomerization. Studies of circular dichroism and intrinsic fluorescence of tryptophan will be conducted to assess the stability of secondary, tertiary and quaternary structures of native and mutant NDK. By fluorescence will be evaluated and intrinsic anisotropy which will provide data of the movement of the structure, allowing inferences about the oligomerization. By circular dichroism, using a thermal and chaotropic denaturation agents, secondary and tertiary structure changes will be evaluated. The functional effect of these mutations on the quaternary structure of LmNDK will be evaluated by cytotoxicity in the presence of eATP and invasion of macrophages in the presence of LmNDK.

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
VIEIRA, PLINIO SALMAZO; CAMPOS BRASIL SOUZA, TATIANA DE ARRUDA; HONORATO, RODRIGO VARGAS; ZANPHORLIN, LETICIA MARIA; SEVERIANO, KELVEN ULISSES; ROCCO, SILVANA APARECIDA; CAVALCANTE DE OLIVEIRA, ARTHUR HENRIQUE; CORDEIRO, ARTUR TORRES; LOPES OLIVEIRA, PAULO SERGIO; DE GIUSEPPE, PRISCILA OLIVEIRA; et al. Pyrrole-indolinone SU11652 targets the nucleoside diphosphate kinase from Leishmania parasites. Biochemical and Biophysical Research Communications, v. 488, n. 3, p. 461-465, . (11/20569-2, 10/03761-4, 07/06755-2, 11/24178-8, 10/51730-0)
VIEIRA, PLINIO SALMAZO; DE GIUSEPPE, PRISCILA OLIVEIRA; CAVALCANTE DE OLIVEIRA, ARTHUR HENRIQUE; MURAKAMI, MARIO TYAGO. The role of the C-terminus and Kpn loop in the quaternary structure stability of nucleoside diphosphate kinase from Leishmania parasites. Journal of Structural Biology, v. 192, n. 3, p. 336-341, . (11/20569-2, 10/03761-4, 07/06755-2, 11/24178-8, 10/51730-0)
VIEIRA, PLINIO S.; DE JESUS SANTOS, ANA P.; DE OLIVEIRA, ARTHUR H. C.. Biophysical Characterization of the Nucleoside Diphosphate Kinase of Leishmania major and Effect of the P95S Mutation. PROTEIN AND PEPTIDE LETTERS, v. 23, n. 2, p. 99-106, . (11/20569-2, 07/06755-2, 10/03761-4)

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