title: Orthocaspases are proteolytically active prokaryotic caspase homologues: the case of Microcystis aeruginosa.
abstract: Caspases are a family of cysteine-dependent proteases known to be involved in the process of programmed cell death in metazoans. Recently, cyanobacteria were also found to contain caspase-like proteins, but their existence has only been identified in silico up to now. Here, we present the first experimental characterisation of a prokaryotic caspase homologue. We have expressed the putative caspase-like gene MaOC1 from the toxic bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806 in Escherichia coli. Kinetic characterisation showed that MaOC1 is an endopeptidase with a preference for arginine in the P1 position and a pH optimum of 7.5. MaOC1 exhibited high catalytic rates with the kcat /KM value for Z-RR-AMC substrate of the order 10(6)  M(-1)  s(-1). In contrast to plant or metazoan caspase-like proteins, whose activity is calcium-dependent or requires dimerisation for activation, MaOC1 was activated by autocatalytic processing after residue Arg219, which separated the catalytic domain and the remaining 55 kDa subunit. The Arg219Ala mutant was resistant to autoprocessing and exhibited no proteolytic activity, confirming that processing of MaOC1 is a prerequisite for its activity. Due to their structural and functional differences to other known caspase-like proteins, we suggest to name these evolutionary primitive proteins orthocaspases.
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000, Ljubljana, Slovenia.
authors.forename: Marina
authors.initials: M
authors.lastname: Klemenčič
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000, Ljubljana, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Novinec
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000, Ljubljana, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Dolinar
mesh_terms: D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000378:metabolism; D007700:Kinetics; D046931:Microcystis / Q000201:enzymology*; D009154:Mutation; D059748:Proteolysis; D016415:Sequence Alignment
chemical_list: D006867:Hydrolases; D020169:Caspases