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.

Highlights

  • Sea ices, an important part of polar oceans, critically influence the productivity of the polar oceans, global energy budgets, and atmosphere-ocean interactions in the Arctic and Antarctic zones [1]

  • The results showed that they both have the same N-terminal sequence (ADATGPGGNLKTGLY) as E495-M, indicating that they were the different forms of protease E495 with different molecular weights (MW)

  • The sequence of E495 has been deduced from its gene sequence, and the domain architecture of E495 precursor has been predicted with the Circular Dichroism (CD)-search service available at NCBI [28]

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Summary

Introduction

An important part of polar oceans, critically influence the productivity of the polar oceans, global energy budgets, and atmosphere-ocean interactions in the Arctic and Antarctic zones [1]. Heterotrophic bacteria and unicellular algae represent the two major groups of sea-ice assemblages [2]. The representative of the M4 family, thermolysin (EC 3.4.24.27) secreted by Bacillus thermoproteolyticus, has been studied in detail and extensively applied for the synthesis of the artificial sweetener aspartame [5]. Most of the M4 metalloproteases studied in detail are the virulence factors of some pathogens [6,7,8]. The M4 metalloproteases are extensively secreted by bacteria from various habitats including soil, sediment and sea ice etc. Compared to study on thermolysin and the metalloproteases which are the virulence factors of some pathogens, study on the characteristics and function of the M4 metalloproteases from other bacteria is rare, especially those from the bacteria derived from deep-sea and polar habitats

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