Heterologous expression and characterisation of microcystinase Dariusz Dziga a, * , Benedykt Wladyka b , Gabriela Zieli  nska b , Jussi Meriluoto c , Marcin Wasylewski d a Department of Plant Physiology and Development, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Kraków, Poland b Department of Analytical Biochemistry, Jagiellonian University, Kraków, Poland c Department of Biosciences, Åbo Akademi University, Turku, Finland d Department of Cell Biology, Jagiellonian University, Kraków, Poland article info Article history: Received 4 September 2011 Received in revised form 8 December 2011 Accepted 17 January 2012 Available online 1 February 2012 Keywords: Microcystin Biodegradation Microcystinase abstract The first enzyme in the microcystin (MC) degradation pathway identified in bacterial strains is coded by mlrA gene and is referred to as microcystinase. To date, there has been no biochemical characterisation of this enzyme. The results presented herein show a successful heterologous expression of MlrA as well as mutational studies, partial puri- fication and biochemical characterisation of the enzyme. The mutation and inhibition study confirmed previous ideas that MlrA is a metalloprotease and allowed to calculate the inhibition parameters. Moreover, the kinetic parameters of MC-LR linearization were measured showing that MlrA exhibits a positive cooperativity towards MC-LR. Further- more, in vitro experiments with Escherichia coli cells expressing MlrA indicated the potency of the heterologous host to eliminate MCs with very high efficiency. This study reports a new approach to the analysis of a microcystin degrading enzyme, extends the knowledge about MC biodegradation and opens broad scope for future study. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The occurrence of cyanotoxins during and after cyano- bacterial blooms has important implications for both recreational use of lakes and reservoirs and drinking water abstraction (van Apeldoorn et al., 2007). The presence of cyanotoxins in water and their accumulation in aquatic organisms create a potential risk to humans exposed to toxins either directly or through the food chain. Hepato- toxic microcystins (MCs), due to their cyclic structure and unusual amino acids composition, are very stable and exhibit resistance to many chemical agents and enzymatic hydrolysis by common proteases (Mazur and Plinski, 2001). A bacterial strain capable of MC degradation was reported by Jones et al. (1994). A subsequent study (Bourne et al., 1996) identified the MC degrading bacterium as a new Sphingomonas species (ACM-3962). The MC degradation pathway was described as a three-step process which involves linearisation of MC molecule, formation of a tet- rapeptide and subsequent hydrolysis of these products catalysed by three enzymes MlrA, MlrB and MlrC, respec- tively. Some further bacteria (mostly belonging to the Proteobacteria family) with degradation activity against MCs were identified in environmental samples (Edwards and Lawton, 2009). The gene cluster involved in the biosynthesis of the enzymes was also characterized (Bourne et al., 2001). By cloning the total genomic DNA of Sphingomonas sp. a 5.8 kb sequence that codes for proteolytic enzymes with activity against MCs was found. Within the sequence four genes were identified: mlrA, mlrB, mlrC and mlrD. The first three genes encode proteins exhibiting the enzymatic activity observed in Sphingomonas sp. (Bourne et al., 1996), whereas the mlrD gene probably encodes a transporter protein. Later sequence analysis (Bourne et al., 2001) described MC degrading enzymes more precisely but many questions remained unanswered. The exact location of the start and * Corresponding author. Tel.: þ48 12 6646540; fax: þ48 12 6646902. E-mail address: dariusz.dziga@uj.edu.pl (D. Dziga). Contents lists available at SciVerse ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon 0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2012.01.001 Toxicon 59 (2012) 578–586