Site directed mutagenesise of l-asparagine as an improveed anti cancer protein with increased l-asnase activity

Samaneh Mirtaheri,1,* Ardeshir hesampour �,2 Fahimeh nemati mansour,3

1. Department of Biotechnology, Faculty of Advanced Sciences & Technology, pharmaceutical sciances Branch, Islamic Azad Uni
2. Department of Biology, Central Tehran Branch, Islamic Azad University,Tehran, Iran

Abstract

Introduction

L-asparaginase (l-asnase) is an fda approved enzyme which is widely used in fight against acute lymphoblastic leukemia and food industry. in view of these facts, rational design of l-asnase mutants with enhanced l-asnase activity would bring about higher effectivity. in this regard, asp96 of this enzyme have previously been proposed to be an important active site amino acid to increase the l-asnase activity. therefore, we have designed and tested several mutant l-asnases to assess their effect on l-asnase activity.

Methods

To this end, the structure of l-asnase and asp was extracted from pdb site at http://www.rcsb.org/pdb/home/home.do and prepared by context software. then, the d96s, d96l, d96i and d96a were introduced using pymol and modeller software. the mutated structures were energy minimized using chimera software. ultimately, using autodock vina software, the structure of wild type and mutated l-asnases were docked against asp.

Results

The structures of l-asnase and asp were successfully obtained, prepared, mutated and docked against each other. the docking results indicated that the d96l (-4.1 kcal/mol) and d96a (-4.5) mutations lead to undesirable decreased asp binding affinity. d96i mutation does not have a tangible effect on binding affinity, while the d96s mutation results in higher asp binding affinity.

Conclusion

Rational design of mutant enzymes with bioinformatics tools could be deemed as an appealing alternative for conventional experimental methods. using these tools would drastically decrease the required time and expenses. using this approach we have designed a more effective mutant l-asnase (d96s) which potentially could replace the currently employed ones.

Keywords

L-asnase, bioinformatics, mutation, stability, molecular docking