Bacteriolytic And Elastolytic Mechanism Of A Novel M23Metalloprotease Pseudoalterin From Deep-sea And Its Function In Ecology And Antimicrobial Therapy

Degradation of organic nitrogen is an important part of global nitrogen cycling. In the ocean, High-Molecular-Weight Organic Nitrogen (HMWON) produced by organisms in the seawater settles and accumulates in the sediment in the form of particulate organic nitrogen (PON). Most of PON will be degraded into dissolved organic nitrogen (DON) by microorganisms, and then participates into the nitrogen cycling through ammonium regeneration, nitrification and denitrification in the sediment. Thus, the recycling of PON in deep-sea sediment would be a non-negligible part of ocean nitrogen cycling. Recent studies indicate that a significant fraction of PON is composed of insoluble amides and organisms living in the sediment. Because of the insoluble properties, collagens and elastins are speculated to be important components of sedimental PON. Elastins are made up of tropoelastin monomers with alternating hydrophobic and hydrophilic domains which are cross-linked complicatedly. Because of the rigid structure, elastins are resistant to most proteases except for a limited number of elastases. Up to now, the reports about elastolytic proteases were mostly focused on elastases from animals. The studies of elastases from microorganisms are relatively fewer and most of bacterial elastases are pathogenic and terrestrial. Thus the elastolytic mechanism of the ocean is unclear. There are abundant elastase-produced bacteria in deep-sea sediment. Elastases from different strains show different elastolytic abilities which imply the diversity of elastases in deep-sea. The peptidoglycans of bacteria living in sediment are also important components of sedimental PON. However, the studies of enzymes from deep sea that could degrade peptidoglycans are limited. Research into the function and mechanism of marine proteases will provide important implications for deep-sea nitrogen cycling and novel proteases development.Pseudoalteromonas sp. CF6-2was isolated from the deep-sea sediment at a water depth of2,441m in the Jiulong methane reef area off the southwest of the island of Taiwan during the South China Sea Open Cruise of R/V Shiyan3. Our previous study showed that P. sp. CF6-2could produce hydrolytic zone on elastin agar plates with the hydrolytic zone diameter/the colony diameter (H/C)=9.0, suggesting that P. sp. CF6-2is a good producer of elastase. Because no elastase from bacteria in Pseudoalteromonas genus has been reported, we speculated that strain CF6-2secrets a novel elastase. In this thesis, the elastase, named pseudoalterin. was purified from the fermentation broth of P. sp. CF6-2and its enzymatic characteristics, gene sequence, degradation mechanisms to elastin and peptidoglycan and application potential were studied. The results are as following:(1) Purification, characteristics and gene cloning of pseudoalterinA protein with a molecular mass of19kDa was purified from the fermentation broth by a DEAE-Sepharose Fast Flow chromatography. which was designated as pseudoalterin. The determination of the substrate specificity showed that pseudoalterin could hardly hydrolyze any protein substrate except elastin. This indicated that it is a strict elastase. Pseudoalterin retained10%activity at5℃and its optimum temperature was25℃. Its thermostability was very low. and70%activity would be lost after it was incubated at35℃for20min. These properties indicated that this protease was a typical cold-adapted enzyme. Pseudoalterin was an alkaline elastase with an optimum pH of9.5. Lower than20%activity could be detected under pH8.0while40%activity was retained at pH10.5. Ca2+and Sr2+markedly increased the enzyme activity of pseudoalterin, and Fe2+severely inhibited the enzyme activity. Zn2+abolished the elastolytic activity completely. Moreover, the activity of pseudoalterin could be inhibited by metalloprotease inhibitors. So pseudoalterin was a Zn2+dependent metallopeptidasc.The full-length gene of pseudoalterin was cloned from P. sp. CF6-2by PCR and thermal asymmetric interlaced PCR (Tail PCR) techniques. The ORF contains1212bp, encoding a precursor of403amino acid residues. The nucleotide sequence was submitted to the GenBankTM with accession number HQ005379. Analysis of the amino acid sequence showed that pseudoalterin is a zinc metalloprotease. Among the characterized proteases, pseudoalterin shows the highest identity (39%) to staphylolysin (M23A subfamily) from Pseudomoiuis aeruginasa PAO1(accession numberAAG05260). The conserved residues in and around the active site of M23 proteases are also found in pseudoalterin. These results all point to pseudoalterin as a novel metalloprotease of the M23A subfamily.Analysis of the genomes of21bacteria isolated from deep-sea sediments and hydrothermal vents, which are in GenBankTM database, indicates that most of these bacteria could secrete one or several M23family proteases. This finding suggests that the M23family proteases may be popular in deep sea sediments and may therefore play an important role in sedimentary PON degradation. However, the properties and functionality of marine M23proteases have not been reported. Pseudoalterin was the first characterized marine elastase in M23family.(2) Elastolytic mechanism of pseudoalterinM23proteases could slowly degrade elastions. However, researches of elastolytic mechanism of M23proteases were limited to the analysis of their cleavage sites on elastin or elastin-like peptides. As a M23A subfamily protease, pseudoalterin could quickly degrade insoluble elastin into soluble peptides. Studying its elastolytic mechanism is helpful for clarifying the degradation mechanism of M23elastases. Microscopic observations indicated that pseudoalterin swelled elastin and separated elastin into filaments, implying that this enzyme initially broke down cross-links between the filaments. Analysis of the cleavage sites of pseudoalterin on bovine elastin and synthetical peptides indicated that pseudoalterin preferentially hydrolyzed elastin cross-linking domains. To further study the elastolytic mechanism of pseudoalterin, scanning electron microscope (SEM) observation of recombinant tropoelastin spherules and bovine elastin degradation process was performed. Based on our biochemical results and SEM observation, the elastolytic mechanism of pseudoalterin was concluded:pseudoalterin successively released filaments, droplets, and spherules from elastic fibers by destroying the cross-links at each structural level. This is the first detailed research of a M23elastase about its elastolytic mechanism.All the proteases in M23family with investigation only cleaved Gly-Xaa bonds in hydrophobic domains in elastin. Therefore, that pseudoalterin hydrolyzed cross-linking domains in elastin before hydrophobic domains represents a new-type elatolytic mechanism in M23family. It also revealed that pseudoalterin is a novel protease in this family. The insight into the novel elastase from the sediment and its mechanism provides important evidences for nitrogen cycling in deep-sea sediment. The research also provides theoretical basis for the development of novel proteases.(3) Comparison of pseudoalterin, myroilysin and pseudolysin on their elastolytic mechanismOf all the elastases from terrestrial bacteria, pseudolysin in M4family from pathogenic bacteria Pseiidomonas aerugonosa was detailed investigated. Myroilysin was the only marine elastase of which the elastin degradation pattern was studied. Myroilysin is an M12family protease from deep-sea sediment bacterium Myroides profundi D25. The detailed elastolytic mechanisms of pseudolysin and myroilysin are still unclear. We studied the elastolytic mechanisms of pseudolysin and myroilysin by biochemical experiments and SEM observation in detail, and compared them with that of pseudoalterin. Although the degradation patterns between myroilysin and pseudolysin were slightly different under light microscope, they both formed cavities at the surface of elastin fibres under SEM. This was completely different from pseudoalterin that separated elastin into filaments firstly. Analysis of the cleavage sites of myroilysin and pseudolysin showed that they foeused on elastin hydrophobic domains but not hydrophilic domains involved in cross-linking, which may explain the different elastolytic patterns among them.Investigation and comparison of the elastolytic mechanism of pseudoalterin. myroilysin and pseudolysin. which are from three distinct families, would be helpful to further understand the elastolytic mechanisms of elastases.(4) The ecological role of the pseudoalterin and its mechanism to absorb and degrade peptidoglycanEnzymes in M23family are all amidases and/or endopeptidases in peptidoglycan degradation. So the peptidoglycans of various marine bacteria were used as substrates to test the antimierobial spectrum of pseudoalterin. It was showed that pseudoalterin lysed both Gram positive and Gram negative bacteria isolated from marine environments, although the activities were not equal because of different structures among genus. Therefore, the bacteriolytic activity of pseudoalterin may have significant ecological role in deep sea and play important roles in the cycling of sedimentary organic matter.Among various kinds of peptidoglycans, the peptidoglycan of Staphylococcus aureus is the favorite substrate of M23proteases. Therefore, the peptidoglycan of S. aureus was chosen to determine the degradation mechanism of pseudoalterin. Our result showed that in addition to hydrolyze the peptidoglycan of S. aureus quickly, pseudoalterin could also adsorb to it and other insoluble polysaccharides, such as chitin, chitose and cellulose. We hypothesized that pseudoalterin could bind to the glycans and degrade the peptides in peptidoglycan degradation.In order to study the peptidoglycan degradation mechanism of pseudoalterin, analysis of cleavage sites on peptidoglycan by pseudoalterin was performed. The result showed that pseudoalterin was both an amidase and an endopeptidase. Except for the glycyl bonds in peptide bridges, the amido bond N-acylmuramoyl-Ala and peptide bonds Ala-Lys and Ala-Glu in peptidoglycan could also be hydrolyzed by pseudoalterin.To gain insights into the structural basis of its adsorption and degradation mechanisms to peptidoglycan, pseudoalterin was crystallized at a resolution of1.5A. Structure analysis showed that a molecule of pseudoalterin is composed of a short α-helix (α1) and9βsheets (β1-β9). The helix α1(104-108) is located in lateral region of the molecule. Five longer antiparallel β sheets:β1(30-35), β2(56-61), β3(64-69), β4(72-78) and β5(119-125), are surrounded by loops at the center, forming a big cleft. Zn2+is located at the upper part of this cleft, indicating that this cleft is the catalytic center. Four shorter antiparallel sheets β6(134-137), β7(140-143), β8(157-160) and β9(164-165) arrange at the top right corner of the pseudoalterin molecule and form a smaller groove near Zn2+, which may interact with substrates. Pseudoalterin is a β protein, just like the other three M23proteases with tertiary structures. However, the structure of pseudoalterin has some difference from those of the other three M23proteases. In addition to the catalytic cleft, pseudoalterin has only one β domain at its top right, but the other proteases have two, one at its top right and the other at the bottom left of the molecule. Moreover, compared to those of the other proteases, the β-sheets at the top right of pseudoalterin are longer and form a wider groove, which may be responsible for peptidoglycan binding.A proposed model of the pseudoalterin-(NAG)4complex was obtained using docking algorithm. The mode indicates that (NAG)4can be easily bound in the groove formed by (36-P9sheets in pseudoalterin. The distances shorter than3.2A between (NAG)4and pseudoalterin amino acid residues were calculated with pymol software. Analysis showed that Asn134, Argl41, Asp143, Tyr157, Glu159and Arg164were likely to form hydrogen bonds with (NAG)4。 Three amino acid residues His133, Asnl54and Arg163located in this region were also taken in our consideration. Mutations of these residues were constructed to verify the key amino acids in the adsorption process. The binding assays revealed that the last20amino acids paly important roles in pseudoalterin adsorption and the mutation of Tyrl57. Glu159, Arg163and Arg164caused dramatically decreases in pseudoalterin adsorption. Therefore, the top-right β domain is confirmed to be responsible for substrate binding. This is the first comprehensive study of an M23protease about its peplidoglycan adsorption and degradation mechanisms, which play important roles in clarifying the bacteriolytic mechanism of M23proteases.(5) The application potential of pseudoalterin and the optimization of its productionNon-antibiotic drugs are becoming an urgent need because of increasing of multiple resistant bacteria. Because lysozymes degrade conserved structures of bacterial peptidoglycan and hardly lead to bacterial resistance, they have great application potential in anti-bacterial therapy. Pseudoalterin may have the promising use in therapy as both an amidase and an endopeptidase to degrade peptidoglycan rapidly. Peptidoglycans of various terrestrial Gram positive and Gram negative bacteria (including methieillin-resistance Staphylococcus aureus, MRSA) were extracted to test the bacteriolytic activity of pseudoalterin. The bacteriolytic effect of pseudoalterin was compared with commercialized lysozyines:mutanolysin and lysostaphin. The results showed that pseudoalterin had lytic activity to most of the gram positive bacteria and the lytic activity to S. aureus was100fold higher than lysostaphin. As a single enzyme, pseudoalterin even had better bacteriolytic effect than complex enzyme mutanolysin to some bacteria. Pseudoalterin had the highest bacteriolytic activity to S. aureus and MRSA, which were both about75%, indicating that MRSA has no resistance to pseudoalterin. The ability of pseudoalterin on killing live cells of S. aureus suggested that pseudoalterin has potential as an anti-bacterial agent.Pseudoalterin was an induced elastase and elastin was indispensable compound in the fermentation medium for pseudoalterin production. However, purified elastin was too expensive to be used in large scale production of pseudoalterin. To reduce the cost for pseudoalterin production, various inducers were tested for their induction effects on pseudoalterin production. The result showed that bovine artery could be used as an inducer to replace elastin without loss in enzyme production. In order to find out the key factors significantly affecting pseudoalterin production of strain CF6-2, the relative significances of variables were investigated using one-factor experiment. The results showed that artery dosage, ferment temperature and ferment time were the greatest important variables for pseudoalterin production. Thus, artery dosage, ferment temperature and ferment time were selected for further optimization using central composite design (CCD). By solving the inverse matrix using Expert-Design software, the optimal values for pseudoalterin production were1.2%for artery,20.17℃for ferment temperature and28.04h for ferment time. Under the optimal conditions mentioned above, the maximum pseudoalterin activity in fermentation broth reached100.02±9.0U/mL. By optimization of the medium composition and the culture conditions using response surface methodology, pseudoalterin activity in fermentation broth was doubled. Our results improved the production of enzyme and greatly reduced the cost of fermentation, which provide a foundation for developing pseudoalterin as an anti-bacterial agent.