Biological diversity of ASNAses: evaluation of enzyme evolution and determination of the crystallographic structure of the ASNaseM

Abstract

Bacterial asparaginases (ASNase) are high molecular weight tetrameric enzymes (~140kDa) used as therapeutic proteins for treatment of acute lymphoblastic leukemia (ALL), since some tumor cell types are dependent on the availability of extracellular L-asparagine. Bacterial ASNase efficiently hydrolyzes aparagine (L-Asn) into aspartic acid (L-Asp) and ammonia, decreasing the source of L-Asn to tumor cells. International pharmaceutical industries produce ASNase from Escherichia coli (EcA) and Erwinia chrysantemi (ErA), but ASNase is not produced by the Brazilian pharmaceutical companies. Additionally, despite being a widely used drug, several side effects are associated with ASNase uses, including immunological reactions, neurotoxicity and coagulation abnormalities. In order to reduce adverse reactions caused by therapy with EcA, PEG-ASNase was developed, which has the advantage of having a longer biological half-life than native ASNase, and yet has lower immunogenicity. Nevertheless, it was demonstrated that there is a cross-reaction between native ASNase and PEG-ASNase, precluding the change from one to the other. The alternative is the replacement with ASNase from Erwinia chrysanthemi, ErA, which causes reduced stimulation of the immune system, in spite of its shorter than EcA biological half-life. However, 33% of patients still present allergic reactions to the formulation. Therefore, the exploration for new sources of ASNases in order to increase its availability as a drug is essential to reduce side effects and to avoid treatment failures due to fluctuations in the production of these biopharmaceuticals. Taking into account the advantages of using microorganisms in bioprocess and the unexplored magnitude of microbial diversity, microorganisms should be considered a target source for new ASNases with improved properties compared with those currently employed in ALL therapy. In this work, we have cloned, expressed and purified an enzyme named Asp1 from Saccharomyces cerevisiae, with high homology with bacterial counterparts (~36% identity and ~54% similarity). However, the enzyme Asp1 has unique structural characteristics as an N-terminal tail containing ~ 50 amino acids. We also standardized methodologies for their expression and purification, and size exclusion chromatography experiments revealed a monomeric enzyme (~ 45 kDa) with elevated asparaginase activity (~110 IU/mg) and presented, as glutaminase-free enzyme, interesting features which would make it less immunogenic enzyme, and have applications in therapy of other cancers. Also, the enzyme ASNaseM presented cytotoxic activity to the neoplastic cells similar to that observed for the bacterial enzyme, suggesting a potential alternative in the treatment of ALL. In this context, the crystallography structure would be of paramount importance to understand the unique structural characteristics of the enzyme. Additionally, it was observed that the N-terminal tail is present only in a very small group of yeasts (~30 species) and preliminary analysis indicates a high variability of the enzymes among the kingdoms of life. In this context, an interesting approach relies in the in silico study of the primary and tertiary/quaternary structure aiming to better understand the ASNase variability in order to classify the different types of enzymes in different families according to evolutive features, possible differences in the catalytic efficiency and structural characteristics. (AU)