title: Characterization of a novel subtilisin-like protease myroicolsin from deep sea bacterium Myroides profundi D25 and molecular insight into its collagenolytic mechanism.
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.
authors.affiliation: From the State Key Laboratory of Microbial Technology.
authors.forename: Li-Yuan
authors.initials: LY
authors.lastname: Ran
authors.forename: Hai-Nan
authors.initials: HN
authors.lastname: Su
authors.forename: Ming-Yang
authors.initials: MY
authors.lastname: Zhou
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Xiu-Lan
authors.initials: XL
authors.lastname: Chen
authors.forename: Bin-Bin
authors.initials: BB
authors.lastname: Xie
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Song
authors.forename: Mei
authors.initials: M
authors.lastname: Shi
authors.forename: Qi-Long
authors.initials: QL
authors.lastname: Qin
authors.forename: Xiuhua
authors.initials: X
authors.lastname: Pang
authors.forename: Bai-Cheng
authors.initials: BC
authors.lastname: Zhou
authors.forename: Yu-Zhong
authors.initials: YZ
authors.lastname: Zhang
authors.forename: Xi-Ying
authors.initials: XY
authors.lastname: Zhang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D041965:Flavobacteriaceae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D012623:Seawater / Q000382:microbiology*; D020860:Subtilisin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014871:Water Microbiology
chemical_list: D001426:Bacterial Proteins; D003094:Collagen; D020860:Subtilisin
keywords: Bacteria; Biodegradation; Collagen; Collagenolytic Mechanism; Deep Sea Sediment; Enzyme Mechanisms; Nitrogen Cycling; Serine Protease; Structural Characteristics; Subtilisin-like Protease