Structural and Functional Characterization of Mature Forms of Metalloprotease E495 from Arctic Sea-Ice Bacterium Pseudoalteromonas sp. SM495

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.

E495是北极海冰细菌假交替单胞菌（Pseudoalteromonas sp.）SM495分泌的丰度最高的蛋白酶。作为嗜热菌蛋白酶家族金属蛋白酶，研究发现E495在SM495菌株的培养物中存在多种活性形式。其中，仅含催化结构域的E495-M与携带催化结构域及1个PPC结构域（PPC domain）的E495-M-C1为两种稳定的成熟形式，而携带催化结构域与2个PPC结构域的E495-M-C1-C2可能为中间产物。相较于E495-M，E495-M-C1对寡肽的亲和力与催化效率基本一致，但对蛋白质的亲和力与催化效率显著更高。将E495来源的PPC结构域构建为谷胱甘肽S-转移酶（GST）融合蛋白进行表达后，实验证实两种重组PPC结构域均具备结合C-藻蓝蛋白与酪蛋白的能力，且PPC1结构域对C-藻蓝蛋白的亲和力高于PPC2结构域。上述结果表明，E495-M-C1中的PPC1结构域可辅助结合蛋白质底物，进而提升酶的催化效率。对PPC结构域开展的定点诱变实验显示，该结构域中保守的极性与芳香族残基D26、D28、Y30、Y/W65在蛋白质结合过程中发挥关键作用。本研究可为北极海冰环境中有机氮的降解机制研究提供新的理论见解。