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Saccharomyces cerevisiae L-asparaginase 1: rational modifications aiming the modulation of kinectic chatacteristics over different substrates

Grant number: 15/20866-8
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): January 01, 2016
Effective date (End): December 31, 2016
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Marcos Antonio de Oliveira
Grantee:Livia Cerf Quintero
Home Institution: Instituto de Biociências (IB-CLP). Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil
Associated research grant:13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical, AP.TEM

Abstract

Acute Lymphoblastic Leukemia (ALL) is a type of haematological malignancy very common in children and teenagers. The ALL neoplastic cell, in contrast with other body cells, do not synthesize the amino acid asparagine (Asn), and the uptake of this amino acid from the extracellular environment it is indispensable the to neoplastic cells survival. In this context, the bacterial L-asparaginases (ASPase) are proteins used in the treatment of ALL, since they are able to deplete this amino acid from the bloodstream, causing the death of neoplastic cells. Despite being widely used in the treatment of ALL, some authors attribute many side effects to the secondary glutaminase activity (GLNase) of these enzymes. However, it was recently shown that in Escherichia coli ASPase (EcA), whose GLNase activity has been decreased or abolished, the appearance of a large decrease in the cytotoxicity of the enzyme on certain types of neoplastic ALL lineages. Works involving determination of the structural and functional analysis of EcA elucidated various amino acids involved in substrate binding and catalysis. Among them, there are Thr residues involved in these processes (Thr12 and Thr89) and site directed mutagenesis studies of ASPases and unrelated enzymes carrying Thr for Ser reveals that this kind of substitution can alters significantly the kinetic parameters of enzyme. Although none systematic investigation involving mutants to both Thr residues or combinatorial mutants was performed to date. Recently, it was also shown that the substitution of Asn24 residue from EcA by moderately increased the ASPase activity. Analyses in databases revealed that Saccharomyces cerevisiae has an enzyme denominated Asp1 with significant homology with EcA (~ 36% identity and ~ 54% similarity) and all the amino acids involved in catalysis are conserved, including Thr (Thr64 and Thr141). However, the Asn24 residue is naturally replaced by Ala (76), which may render to enzyme a higher activity over Asn, as recently demonstrated in EcA. The objectives of this work lie in the functional and structural characterization of recombinant enzymes Asp1T64S, Asp1T141S, Asp1T64S/T141S and Asp1K247S, aiming a better understanding of the dual gluaminase and asparaginase activities of the enzyme. First, the expression and purification conditions will be standardized and the structural characterization will be perfomed by SDS PAGE, size exclusion chromatography (SEC) and circular dichroism spectroscopy (CD). The evaluation of activities over Asn and Gln will be performed using the Nessler's method and by steady state kinetics following NADH oxidation of assay. We believe that the results of this project are important not only for characterization of the S. cerevisiae enzyme which may be an promise alternative to bacterial enzymes, but also will provide a better understanding of amino acids involved in ASPases catalysis and substrate binding, since the amino acids investigated are widely conserved in ASPases from various microorganisms.