Characterization of a Novel Subtilisin-like Protease Myroicolsin from Deep Sea Bacterium Myroides profundi D25 and Molecular Insight into Its Collagenolytic Mechanism |
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Authors: | Li-Yuan Ran Hai-Nan Su Ming-Yang Zhou Lei Wang Xiu-Lan Chen Bin-Bin Xie Xiao-Yan Song Mei Shi Qi-Long Qin Xiuhua Pang Bai-Cheng Zhou Yu-Zhong Zhang Xi-Ying Zhang |
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Institution: | From the ‡State Key Laboratory of Microbial Technology.;the §Marine Biotechnology Research Center, and ;the ¶Collaborative Innovation Center of Deep Sea Biology, Shandong University, Jinan 250100, China |
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Abstract: | Collagen is an insoluble protein that widely distributes in the extracellular matrix of marine animals. Collagen degradation is an important step in the marine nitrogen cycle. However, the mechanism of marine collagen degradation is still largely unknown. Here, a novel subtilisin-like collagenolytic protease, myroicolsin, which is secreted by the deep sea bacterium Myroides profundi D25, was purified and characterized, and its collagenolytic mechanism was studied. Myroicolsin displays low identity (<30%) to previously characterized subtilisin-like proteases, and it contains a novel domain structure. Protein truncation indicated that the Pro secretion system C-terminal sorting domain in the precursor protein is involved in the cleavage of the N-propeptide, and the linker is required for protein folding during myroicolsin maturation. The C-terminal β-jelly roll domain did not bind insoluble collagen fiber, suggesting that myroicolsin may degrade collagen without the assistance of a collagen-binding domain. Myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. The favored residue at the P1 site was basic arginine. Scanning electron microscopy and atomic force microscopy, together with biochemical analyses, confirmed that collagen fiber degradation by myroicolsin begins with the hydrolysis of proteoglycans and telopeptides in collagen fibers and fibrils. Myroicolsin showed strikingly different cleavage patterns between native and denatured collagens. A collagen degradation model of myroicolsin was proposed based on our results. Our study provides molecular insight into the collagen degradation mechanism and structural characterization of a subtilisin-like collagenolytic protease secreted by a deep sea bacterium, shedding light on the degradation mechanism of deep sea sedimentary organic nitrogen. |
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Keywords: | Bacteria Biodegradation Collagen Enzyme Mechanisms Serine Protease Collagenolytic Mechanism Deep Sea Sediment Nitrogen Cycling Structural Characteristics Subtilisin-like Protease |
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