title: Structural and functional characterization of mature forms of metalloprotease E495 from Arctic sea-ice bacterium Pseudoalteromonas sp. SM495.
abstract: E495 is the most abundant protease secreted by the Arctic sea-ice bacterium Pseudoalteromonas sp. SM495. As a thermolysin family metalloprotease, E495 was found to have multiple active forms in the culture of strain SM495. E495-M (containing only the catalytic domain) and E495-M-C1 (containing the catalytic domain and one PPC domain) were two stable mature forms, and E495-M-C1-C2 (containing the catalytic domain and two PPC domains) might be an intermediate. Compared to E495-M, E495-M-C1 had similar affinity and catalytic efficiency to oligopeptides, but higher affinity and catalytic efficiency to proteins. The PPC domains from E495 were expressed as GST-fused proteins. Both of the recombinant PPC domains were shown to have binding ability to proteins C-phycocyanin and casein, and domain PPC1 had higher affinity to C-phycocyanin 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 showed that the conserved polar and aromatic residues, D26, D28, Y30, Y/W65, in the PPC domains played key roles in protein binding. Our study may shed light on the mechanism of organic nitrogen degradation in the Arctic sea ice.
authors.affiliation: State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan, China.
authors.forename: Hai-Lun
authors.initials: HL
authors.lastname: He
authors.forename: Jun
authors.initials: J
authors.lastname: Guo
authors.forename: Xiu-Lan
authors.initials: XL
authors.lastname: Chen
authors.forename: Bin-Bin
authors.initials: BB
authors.lastname: Xie
authors.forename: Xi-Ying
authors.initials: XY
authors.lastname: Zhang
authors.forename: Yong
authors.initials: Y
authors.lastname: Yu
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.forename: Bai-Cheng
authors.initials: BC
authors.lastname: Zhou
authors.forename: Yu-Zhong
authors.initials: YZ
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004795:Enzyme Stability; D007700:Kinetics; D045726:Metalloproteases / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010798:Phycocyanin / Q000378:metabolism; D011485:Protein Binding; D031161:Pseudoalteromonas / Q000201:enzymology; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
chemical_list: D010798:Phycocyanin; D045726:Metalloproteases