The entry reviews the discovery, properties, and functional activities of new bacterial enzymes, proteases grimelysin (ECP 32) of Serratia grimesii and protealysin of Serratia proteamaculans, characterized by both a highly specific “actinase” activity and their ability to stimulate bacterial invasion. Grimelysin cleaves the only one polypeptide bond Gly42-Val43 in actin. This bond is not cleaved by any other proteases and leads to a reversible loss of actin polymerization. Similar properties were characteristic for another bacterial protease, protealysin. These properties made grimelysin and protealysin a unique tool to study the functional properties of actin. Furthermore, bacteria Serratia spp. producing grimelysin/protealysin invade eukaryotic cells, and the recombinant Escherichia coli expressing the grimelysin or protealysins gene become invasive. Being an intracellular enzyme, grimelysin/protealysin can be delivered by bacteria to eukaryotic cells. These data indicate that the protease is a virulence factor, and actin can be a target for the protease upon its translocation into the host cell.
Grimelysin (ECP 32), discovered, purified and initially characterized as protease ECP 32 , was later shown to be identical to grimelysin . Therefore, the properties of the enzyme identified for ECP 32 could be applied to grimelysin. However, here we retain the name grimelysin (ECP 32) and ECP-cleaved actin to comply with the published data where the protease was named ECP 32. Grimelysin (ECP 32), purified from a bacterial extract using sequential chromatography steps, is a single 32 kDa polypeptide, whose N-terminal sequence was determined to be AKTSSAGVVIRDIFL . The optimum of the protease activity was observed in the range of pH 7–8 when actin and melittin were used as substrates . The proteolytic activity increased with increasing ionic strength: in 50–100 mM NaCl the activity of grimelysin (ECP 32) towards melittin was shown to be nearly twice higher than in a low ionic strength solution . It was also enhanced in the presence of millimolar ATP concentrations, though hydrolysis of melittin was not accompanied by ATP hydrolysis at a rate comparable with the cleavage rate. This implies that protease grimelysin (ECP 32) is not an ATP-dependent enzyme , which is important for the experiments involving actin because actin contains ATP as a tightly-bound nucleotide. The protease activity is inhibited by EDTA, EGTA, o-phenanthroline and zincone, and the EDTA-inactivated enzyme can be reactivated by cobalt, nickel and zinc ions . Based on these data, grimelysin (ECP 32) was classified as a neutral metalloproteinase (EC 3.4.24) .
Limited proteolysis of skeletal muscle actin between Gly-42 and Val-43  was observed at enzyme: substrate mass ratios of 1:25 to 1:3000 . Two more sites, between Ala-29 and Val-30 and between Ser-33 and Ile-34, were cleaved by ECP 32 in heat- or EDTA-inactivated actin, apparently due to conformational changes around residues 28–34 buried in intact actin . Besides actin, only melittin , histone H5, bacterial DNA-binding protein HU and chaperone DnaK  were found to be protease substrates. In agreement with this high substrate specificity, ECP 32 did not hydrolyze tropomyosin, troponin, α-actinin, casein, histone H2B, ovalbumin, bovine serum IgG, bovine serum albumin, bovine pancreatic ribonuclease A, trypsin, human heat shock protein HSP70, chicken egg lysozyme  insulin , DNAse I , gelsolin  and profilin . The amino acid residues recognized by grimelysin (ECP 32) in actin and melittin are hydrophobic. This specificity is characteristic for thermolysin-like metalloproteinases . However, high specificity of the enzyme seems to be determined predominantly by conformation at the actin cleavage site rather than its primary structure.
Grimelysin was obtained as a recombinant protein. This has been achieved by cloning the putative gene encoding grimelysin in S. grimesii A2 and in the reference S. grimesii 30063  using published protealysin sequences identified in S. proteamaculans . Grimelysin shared all properties characteristic for ECP 32 including a molecular weight of 32 kDa, an N-terminal 14 amino acid sequence, optimum activity in the range of pH 7–8 and inhibition with o-phenanthroline and EGTA .
Protealysin is a neutral zink-containing metalloprotease of Serratia proteamaculans. The protealysin gene was cloned from a genomic library of S. proteamaculans strain 94 isolated from spoiled meat. This protein was expressed in Escherichia coli and purified as described earlier . Similarly to other thermolysin-like proteases , protealysin is synthesized as a precursor containing a propeptide of about 50 amino acids that is removed during formation of mature active protein . The propeptide is much shorter than the propeptides of the thermolysin-like proteases and has no significant structural similarity to the propeptides of most thermolysin-like proteases . A similar propeptide of 50 amino acids was also detected in the primary structure of the recombinant grimelysin. According to SDS-electrophoresis, recombinant proteins with or without propeptide had an apparent molecular weight of 37 and 32 kDa, respectively .
The molecular weight of the active recombinant protealysin 32 kDa and the N-terminal amino acid sequence AKTSTGGEVI are identical to those of grimelysin . The optimal pH for azocasein hydrolysis is 7, and protealysin is completely inhibited by o-phenanthroline , i.e., has the same properties as grimelysin . Protealysin and grimelysin (ECP 32) are also similar in their unique property of being able to digest actin specifically .