Heterologous expression, purification and characterization of enzymes involved in the biosynthetic pathway of the cyanobacterial neurotoxin anatoxin-a(s)

Abstract

Sphaerospermopsis torques-reginae is a planktonic cyanobacterial species with broad distribution in several Brazilian aquatic environments. Some strains of this species are known to produce the neurotoxic organophosphate anatoxin-a(s) (ANS), which is a powerful irreversible inhibitor of the acetylcholinesterase,enzyme, that acts in the peripheral nervous system of mammals. The ANS structure was already elucidated, however, its biosynthetic pathway is still unknown. The sequencing and assembly of the S. torques-reginae ITEP-024 genome (ANS-producing Brazilian strain) by our research group allowed to detect a gene cluster potentially involved in the ANS production. Thus, this doctoral project aims to overexpress, purify and characterize key enzymes of ANS biosynthesis, and test their activities in order to prove the proposed biosynthetic pathway. For this, the genes encoding the enzymes will be synthetized and inserted in the expression vector for Escherichia coli. The enzymes purification will be performed by affinity and molecular exclusion chromatography and their activities will be tested to prove the formation of the expected intermediates of the ANS biosynthesis. The target enzymes will still have their tridimensional structure determined by X-Ray diffraction. The intermediates production will be verified by chemical analyses using mass spectrometry. This study will contribute to the elucidation of the ANS biosynthetic pathway, which may generate a patent, and also enable the development of rapid and sensitive methods for the detection and monitoring of this toxin in freshwaters used for public supply, as well as offers potential for developing new drugs. (AU)