Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, at 1.4 Å resolution: Structural adaptations to cold and functional analysis of a laundry detergent enzyme

Abstract

Enzymes isolated from organisms found in cold habitats generally exhibit higher catalytic activity at low temperatures than their mesophilic homologs and are therefore known as cold-active enzymes. Cold-active proteases are very useful in a variety of biotechnological applications, particularly as active ingredients in laundry and dishwashing detergents, where they provide strong protein-degrading activity in cold water. We identified a cold-active protease (Pro21717) from a psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, and determined the crystal structure of its catalytic domain (CD) at a resolution of 1.4 Å. The Pro21717-CD structure shows a conserved subtilisin-like fold with a typical catalytic triad (Asp185, His244, and Ser425) and contains four calcium ions and three disulfide bonds. Interestingly, we observed an unexpected electron density at the substrate-binding site from a co-purified peptide. Although the sequence of this peptide is unknown, analysis of the peptide-complexed structure nonetheless provides some indication of the substrate recognition and binding mode of Pro21717. Moreover, various parameters, including a wide substrate pocket size, an abundant active-site loop content, and a flexible structure provide potential explanations for the cold-adapted properties of Pro21717. In conclusion, this is first structural characterization of a cold-adapted subtilisin-like protease, and these findings provide a structural and functional basis for industrial applications of Pro21717 as a cold-active laundry or dishwashing detergent enzyme.

Proteases from the marine bacteria in the genus Pseudoalteromonas: diversity, characteristics, ecological roles, and application potentials

“…The catalytic domain of Pro21717 shows a conserved subtilisinlike fold with a typical catalytic triad (Asp185, His244, and Ser425) and contains four calcium ions and three disulfide bonds. Pro21717 has a wide substrate pocket size, an abundant active-site loop content, and a flexible structure that provide potential explanations for the cold-adapted properties of Pro21717 (Park et al 2018). As subtilisin-like proteases, the structures of the catalytic domains of MCP-01, Apa1, and Pro21717 are similar (Fig.…”

“…1): the psychrophilic alkaline serine protease Apa1 from the Antarctic psychrotroph Pseudoalteromonas sp. AS-11 (Dong et al 2005) and the cold-active protease Pro21717 from the psychrophilic bacterium, P. arctica PAMC 21717 (Park et al 2018). The structure of Pro21717 was analyzed for its cold-adapted properties.…”

“…SM9913 (Ran et al 2013); Apa1, P. sp. AS-11 (Dong et al 2005); Pro21717, P. arctica PAMC 21717 (Park et al 2018) containing a catalytic domain and two PPC domains. Compared to mesophilic subtilisin Carlsberg, MCP-03 has higher activity at low temperatures, lower optimum temperature, and higher thermolability.…”

Adaptation, production, and biotechnological potential of cold-adapted proteases from psychrophiles and psychrotrophs: recent overview

“… Serine protease PMSF and AEBSF Ca 2+ and Mn 2+ 35/9 [ 11 ] Pseudomonas aeruginosa Serine protease PMSF and Ag + Mg 2+ , K + , Ca 2+ , Ba 2+ , and Zn 2+ 25/10 [ 21 ] Pseudoalteromonas sp. Serine protease PMSF, SDS, and H 2 O 2 , Nm 25–35/8–9 [ 16 ] Pseudoalteromonas arctica Subtilisin-like protease Linear alkylbenzene sulfonate (LAS) and SDS Ca 2+ 30/9.0 [ 64 ] Pseudomonas lundensis Metalloprotease EDTA, EGTA, Cu 2+ , Fe 3+ , Al 3+ , Fe 2+ , Mn 2+ , Al 3+ , and Co 2+ Na + , K + , and Li + 30/10.4 [ 19 ] Serratia marcescens Metalloprotease EDTA, MnCl 2 , CaCl 2 , CoSo 4 , HgCl 2 , and Na 2 Nm 40/8 [ 27 ] Sporobolomyces roseus Aspartic protease 2-Mercaptoethanol, dithiothreitol, SDS, and Pepstatin A Nm 50/4 [ 31 ] Stenotrophomonas sp. Alkaline protease Zn 2+ , Cu 2+ , and Co 2+ Mg 2+ , Mn 2+ , and Ca 2+ 15/10 [ 28 ] Stenotrophomonas maltophilia Alkaline protease Co 2+ Cu 2+ , Cr 2+ ...…”

“…was heterologously expressed in E. coli , and recombinant A03Pep1 showed characteristics suitable for industrial applications [ 18 ]. Park et al [ 64 ] cloned the pro21717 gene encoding the psychrophilic serine protease (Pro21717) from Pseudoalteromonas arctica , and the recombinant Pro21717-CD exhibited higher activity at alkaline pH and low temperature. Moreover, Pro21717-CD showed stability against various chemicals and detergent surfactants, making it a valuable product for commercial detergent formulations.…”