Hydrolysis of Insoluble Collagen by Deseasin MCP-01 from Deep-sea Pseudoalteromonas sp. SM9913

Abstract

Collagens are the most abundant proteins in marine animals and their degradation is important for the recycling of marine nitrogen. However, it is rather unclear how marine collagens are degraded because few marine collagenolytic proteases are studied in detail. Deseasins are a new type of multidomain subtilases. Here, the collagenolytic activity of deseasin MCP-01, the type example of deseasins, was studied. MCP-01 had broad substrate specificity to various type collagens from terrestrial and marine animals. It completely decomposed insoluble collagen into soluble peptides and amino acids, and was more prone to degrade marine collagen than terrestrial collagen. Thirtyseven cleavage sites of MCP-01 on bovine collagen chains were elucidated, showing the cleavage is various but specific. As the main extracellular cold-adapted protease from deepsea bacterium Pseudoalteromonas sp. SM9913, MCP-01 displayed high activity at low temperature and alkaline range. Our data also showed that the C-terminal polycystic kidney disease (PKD) domain of MCP-01 was able to bind insoluble collagen and facilitate the insoluble collagen digestion by MCP-01. Sitedirected mutagenesis demonstrated that Trp-36 of the PKD domain played a key role in its binding to insoluble collagen. It is the first time that the structure and function of a marine collagenolytic protease, deseasin MCP-01, has been studied in detail. Moreover, the PKD domain was experimentally proven to bind to insoluble protein for the first time. These results imply that MCP-01 would play an important role in the degradation of deep-sea sedimentary particulate organic nitrogen.Collagens, which distribute in skin, scale, bone, tendon, teeth, and blood vessels are the major protein constituents of the extracellular matrix and the most abundant proteins in all higher organisms including marine animals. It is an important fraction of marine organic nitrogen, and its degradation is important for marine nitrogen recycling.Because the tightly coiled triple helical collagen molecule assembles into water-insoluble fibers, collagens are resistant to most proteases except for a limited number of collagenolytic proteases. The mechanism of marine collagen degradation in marine nitrogen recycling is rather unclear because there are only several reports about marine collagenolytic enzyme-producing bacteria and collagenases (1-5). Collagenolytic proteases include metalloproteinases, serine proteases, and other proteases. Of all the collagenolytic proteases, mammalian collagenases, which are mammalian matrix metalloproteinases have been investigated in more detail than collagenolytic proteases from bacteria (6). Studies on the collagenolytic proteases from marine bacteria are rather minor and preliminary. Some marine collagenolytic enzyme-producing bacteria have been reported (1, 5). The collagenase from marine Vibro B-30 was purified and primarily characterized, which was reported at 1978 and 1980 (2-4). Since then, there has been no report on the characterization of marine bacterial col...

Characterization of a New S8 serine Protease from Marine Sedimentary Photobacterium sp. A5–7 and the Function of Its Protease-Associated Domain

“…However, Ca 2+ only had a little effect on P57 activity. Previous reports showed that Mn 2+ did not affect the enzyme activity of subtilase significantly (Kurata et al, 2007) or slightly inhibited the enzyme activity of subtilase (Zhao et al, 2008; Ran et al, 2014). Our result showed that 8 mM Mn 2+ significantly increased P57 activity.…”

“…SM9913 (Zhao et al, 2008; Ran et al, 2013), the thermostable protease from Geobacillus collagenovorans MO-1 (Okamoto et al, 2001), AcpII from Alkalimonas collagenimarina AC40 (Kurata et al, 2010), and myroicolsin from Myroides profundi D25 (Ran et al, 2014). P57 has collagenolytic activity and its PA domain has collagen-binding ability, indicating that P57 is an S8 serine collagenolytic protease.…”

“…Metal ions have different effects on the activity of different enzymes. It was reported that Ca 2+ could increase the enzyme activity or the thermal stability of some subtilases (Kurata et al, 2007, 2010; Zhao et al, 2008; Ran et al, 2014). However, Ca 2+ only had a little effect on P57 activity.…”

“…Circular dichroism (CD) spectra of the purified PA-EGFP and its mutants F349A-EGFP and Y432A-EGFP in 50 mM Tris-HCl buffer (0.1 mg/mL) were measured on a Jasco J810 spectropolarimeter (Japan) according to the method described by Zhao et al (2008).…”

Integrated metatranscriptomic and metagenomic analyses of stratified microbial assemblages in the open ocean

“…Polycystic kidney disease domains are mostly present on the cell surface, and are involved in protein-protein interactions. Particularly, polycystic kidney disease domains were found predominant in archaeal surface layer proteins that were thought to protect the cell from extreme environments (Jing et al, 2002), or in exported proteins of marine heterotrophic bacteria that may be involved in the binding and degradation of extracellular polymers (carbohydrate and protein) (Zhao et al, 2008). The taxonomic origins and prevalence in community transcriptomes of these polycystic kidney disease domain-containing hypothetical genes now render them reasonable targets for future functional characterizations in planktonic marine Crenarchaea.…”