Multiple Mature Forms Of Metalloprotease E495 In Thermolysin Family Secreted By Sea Ice Bacteria Pseudoalteromonas Sp. SM495 And Function Of Its C-terminal PPC Domain

Sea ices, an important part of polaroceans, critically influence the productivity of the polar oceans. Pseudoalteromonas sp. SM495 is a protease-secreting bacterium isolated from the Arctic sea ice. Vibriolysin E495, the most abundant protease secreted by strain SM495, is a cold-adapted metalloprotease of the thermolysin family. As a thermolysin family metalloprotease, E495 was first found to have three mature forms in this study. That is, E495 (34 kDa) containing only the catalytic domain, E495-M-C1 (45 kDa) containing the catalytic domain and one Pre-Peptide C-terminal domain (PPC), E495-M-C1-C2 (60 kDa) containing the catalytic domain and two PPC domains. We tried to purify the three mature forms, but only E495-M and E495-M-C1 were obtained. Compared to E495-M, E495-M-C1 had similar affinity and catalytic efficiency to low molecular weight oligopeptides, but higher affinity and catalytic efficiency to proteins, such as C-phycocyanin (CPC), allophycocyanin (APC) andα-casein. These results indicated that PPC must have its unique function in substrate catalytic mechanism. The PPC domains from E495 were expressed as GST-fused proteins. "Pull-down" affinity head interaction and Surface Plasmon Resonance (SPR) analyses showed that the recombinant PPC domains had binding ability to high molecular weight proteins C-phycocyanin (CPC) and casein. Binding kinetic constants analysis by SPR indicated that domain PPC1 had higher affinity to CPC than domain PPC2. These results indicated that the domain PPC1 in E495-M-C1 could be helpful in binding protein substrate, and therefore improving the catalytic efficiency. Site-directed mutagenesis on the PPC domains was performed to analyze the key amino acid residues in protein substrate binding. The results showed that the conserved polar and aromatic residues, D26, D28, Y30, Y/W65, in the PPC domains played key roles in protein binding. Tyrosine (Y)/Trypsin (W), which contains huge an aromatic group and helps to maintain the interaction by forming hydrogen bonds and hydrophobic force. Aspartate (D) might interact with the peptide ligands via making a direct salt bridge. Taken together, our results suggest that the multiple mature forms of E495 may be a strategy of strain SM495 to efficiently degrade the proteins in sea ice. Our study may shed light on the mechanism of organic nitrogen degradation in the Arctic sea ice.