Taxonomic Identification Of Arctic Marine Bacteria And Study On The Structure And Catalytic Mechanism Of Polysaccharide Lyase Family 6 Alginate Lyase

The ocean is the largest ecosystem on the earth,with features such as low temperature,high pressure,high salt,low light and oligotrophs.The ocean is rich in biological resources.As an important member of marine life,microorganisms have the characteristics adapting to the marine.environment,such as halophilic or salt-tolerant,high-pressure-resistant,psychrophilic,and oligotrophic.As an extreme marine environment,the Arctic areas contain unique microbial resources that are compatible with their environment,and have great significance and potential for resource development.Brown algae is an important primary productivity in marine ecosystems.Alginate is the major polysaccharide produced by brown algae and can account for 40%of the dry weight of the algae.Alginate is formed by the 1,4-glycosidic bond of two kinds of residues,?-D-mannuronic acid and a-L-guluronic acid,in a variety of ways.Alginate lyase,a kind of polysaccharide lyase,is an important enzyme in nature.It is mostly produced by marine bacteria and fungi.It is directly involved in the degradation of alginate and is closely related to the organic carbon cycle in marine ecosystems.Alginate lyase cleaves 1,4-glycosidic bonds between alginate monomers through the?-elimination mechanism.As a result,the polymer is degraded into a series of oligosaccharide chains of different lengths and unsaturated double bonds form at the C4 and C5 positions of the terminal six-membered rings of the product.Currently,alginate lyases are distributed in the polysaccharide lyase families PL5,-6,-7,-14,-15,-17 and-18(http://www.cazy.org/fam/acc_PL.html).Alginate lyase has a wide range of uses and is mainly used for the structural analysis of alginate,preparation ofprotoplasts of brown algae and preparation of functional alginate oligosaccharides.In addition,some new uses of alginate lyase are also under development,such as the treatment of diseases caused by Pseudomonas aeruginosa,and producing biofuels from brown algae.Therefore,the study on the character,structure and catalytic mechanism of alginate lyase can not only help to further reveal the degradation mechanism of marine brown algae polysaccharides,but also lay a foundation for its application in the field of biotechnology.In this paper,we first identified a strain isolated from the sample of Arctic marine sediments as a novel species,enriching the library of marine microbial strains.Next,we found a PL6 family alginate lyase gene alyGC from a marine bacterium Paraglaciecola chathamensis S18K6T that has been annotated with gene functions in the laboratory.Then,we performed sequence analysis and heterologous expression of the alyGC-encoding protein AlyGC,analyzed the biochemical properties of the recombinant enzyme,solved its protein structure,and studied the function of the C-terminal domain.Finally,through the analysis of its complex structure with the substrate combined with biochemical verification,the catalytic mechanism of the PL6 family alginate lyase AlyGC was clarified.(1)Study on the Taxonomic Identification of the Arctic Novel Bacterial Species SM1214TThe Arctic is one of the most extreme environments and possesses the important microbial resources on earth.However,its extreme environmental conditions have increased the difficulty of studying marine microorganisms in the Arctic,resulting in many new strains with potential applications that have not yet been discovered and utilized.Strain SM1214T isolated from the Arctic marine sediment sample by our laboratory is suspected to be a representive of a novel species.Analyses the morphological,physiological,biochemical,chemical and molecular characters of strain SM1214T by the taxonomic methods showed that cells are Gram-reaction-negative,aerobic,non-flagellated rods(approx.0.2-0.4 ?m in width and 0.5-2 ?m in length).Gliding motility is not observed.Colonies are circular(0.5-1.5 mm in diameter),orange and convex with smooth surfaces.Growth occurs at 10-30?(optimum 25?),with 0.5-5%(w/v)NaCl(optimum 2%)and at pH 6.0-8.0(optimum at pH 7.0).The strain is catalase-and oxidase-positive,hydrolyses casein and esculin but not DNA,elastin,starch or Tween 80.The major cellular fatty acids are iso-C15:0,iso-C17:0 3-OH,iso-C15:1 G,C15:0,sunummed feature 3(C16:1?7c and/or iso-C15:0 2-OH),anteiso-C15:0 and C17:0 2-OH.The genomic DNA G+C content of the type strain is 35.4 mol%.The 16S rRNA gene sequence analysis showed that the 16S rRNA gene sequence of SM1214T has the highest similarity with the strain of the genus Subsaxibacter(96.7%).In the phylogenetic tree constructed based on the 16S rRNA gene sequence,this strain also forms a separate internal branch within the genus Subsaxibacter,clustering with the Subsaxibacter broadyi P7T.It itdicates that the strain SM1214T represents a novel species in the genus Subsaxibacter of the family Flavobacteriaceae of Bacteroidetes,and the novel species is named Subsaxibacter arcticus sp.nov.This is the second species of this genus.The acquisition of new strains has enhanced the understanding of the diversity of marine microbes in the Arctic Sea,laying a foundation for further research and development of marine bacteria and their enzymatic resources.(2)Heterologous Expression and Characterization of Marine Bacterial PL6 Family Alginate Lyase AlyGCAmong the seven polysaccharide lyase families into which alginate lyases are distributed,there are few studies on the PL6 alginate lyases,and no structure and catalytic mechanism of the PL6 alginate lyases have been reported so far.The marine bacterium Paraglaciecola chathamensis S18K6T is a marine strain that has been whole genome sequenced.By annotating gene functions,we discovered that this strain contains a gene alyGC that may encode a PL6 family alginate lyase.We analyzed the sequence of the alyGC-encoding protein AlyGC.Sequence analysis showed that AlyGC consists of two domains,including an N-terminal catalytic domain(NTD)and a functionally unknown C-terminal domain(CTD).We heterologously expressed and purified the protein AlyGC without the signal peptide,and studied the biochemical characters of the recombinant AlyGC.We found that AlyGC has the activity of degrading alginate and found that it has the highest degradation activity on polyguluronate(PG),but it cannot degrade polymannuronic acid(PM).The optimum temperature of AlyGC enzyme reaction is 30?,the optimm pH is 7.0,and the enzyme activity is highest without NaCl It is an exotype enzyme releasing only the monosaccharide products.Gel filtration chromatography and dynamic light scattering experiments showed that AlyGC exists as a dimer in the solution.(3)Analysis of the Structure of Marine Bacterial PL6 Family Alginate Lyase AlyGC and the Function of Its C-terminal DomainThe protein structure of the PL6 family alginate lyase is not yet known,which hinders the study of the function and mechanism of the PL6 family alginate lyase.In this study,we crystallized wild-type and selenomethionine(SeMet)labeled AlyGC proteins and solved the crystal structure of AlyGC.There are four AlyGC molecules in one asymmetrical unit of AlyGC crystal,and there is a close interaction between the two C-terminal domains(CTDs)of two molecules,forming the interface of the dimer.In an AlyGC molecule,both the NTD and the CTD adopt the ?-helix fold;NTD and CTD are linked by a linker to form a tandem ?-helix.The NTD of AlyGC binds a metal ion,which is detected as Ca2+ by inductively coupled plasma optical emission spectrometry,forming a catalytic center with Ca2+ as the core.The CTD main structure of AlyGC does not participate in the formation of the catalytic center.Sequence analysis shows that CTD does not have homologous sequences of known functions,and is present in many PL6 family alginate lyases.In order to reveal the function of the CTD in AlyGC,we performed a structural analysis and biochemical assays.Structural analysis shows that a loop on the CTD extends into the catalytic center.The two amino acid residues D631 and S633 located on the CTD's loop extending into the catalytic center are close to the active center.Mutations of D631A and S633A caused a significant decrease in AlyGC enzyme activity,while the CTD deletion mutant,ACTD,almost lost all the activity.Moreover,the CTD deletion changed AlyGC from a dimer to a monmer in the solution.On the basis of the structural analysis and biochemical results,we considered that the CTD plays an important role in the maintenance of protein dimeric formation and enzymatic activity of AlyGC.(4)The Catalytic Mechanism of Marine Bacterial PL6 Family Alginate Lyase AlyGCThe catalytic mechanism of the PL6 alginate lyase has not been studied so far.To investigate the catalytic mechanism of AlyGC,we obtained the crystal of AlyGC in complex with tetramannuronate(R241A-M4)and solved its structure.R241A-M4 also exists as a dimer in the solution The R241A-M4 dimer contains two catalytic centers,each of which binds one Ca2+.Only one of the two active centers in the R241A-M4 structure binds the substrate M4,in which the carboxyl group of +1 mannuronate interacts with Ca2+.When the Ca2+-coordinated amino acids were mutated to Ala or Ca2+ was chelated with EDTA,AlyGC lost its activity.When Ca2+was added to the apo-AlyGC,the enzyme actiyity recovered.Moreover,the function of Ca2+ cannot be replaced by other divalent metal ions such as Mg2+ and Mn2+.These results showed that Ca2+ is necessary for AlyGC activity.The structural superposition of R241A-M4 and wild-type AlyGC showed that the structure of R241A-M4 dimer has a significant conformational change.Compared with WT AlyGC,the entrance of the active center a that binds the M4 is enlarged and that of the active center P without substrate is smaller(the polysaccharide chain is inaccessible).Based on this,we considered that in the catalytic process of AlyGC,the catalysis of the two catalytic centers may not proceed simultaneously.Based on structural analysis and sequence alignment,we also mutated many amino acid residues that may participate in the catalysis.According to the mutation results,we identified the important amino acid residues involved in substrate binding and catalysis in AlyGC,where Arg241 and Lys220 are the catalytic acid and base,respectively,of the reaction.Combining the structural analysis and biochemical experiment results,we finally proposed the molecular mechanism of the degradation of alginate of AlyGC.AlyGC adopts the metal-ion assisted catalytic mechanism using the Ca2+ to neutralize the negative charge of the carboxyl groiup of the substrate.This is the first report of the catalytic mechanism of the PL6 alginate lyase.In this paper,we first identified a marine microbial strain,and then studied the biochemical characters,crystal structures and catalytik mechanism of the PL6 alginate lyase AlyGC in detail.The results will enrich our understanding of alginate lyase and lay a foundation for better development and utilization of marine microbial resources and marine enzyme resources.