| Alginate is synthesized as a cell wall component by brown algae and also as an exopolysaccharide by certain bacteria. This polysacchatide is a natural liner polysaccharide composed of (1,4)-linked β-D-mannuronate(M) and its C5epimer, a-L-guluronate (G). These uronic acids are arranged in block structures which may be homopolymeric M block (polyM), and homopolymeric G block (polyG), and heteropolymeric blocks with random arragement of both monomers (polyMG) Alginate lyase catalyze the degration of polysaccharides by β-elimination reaction. Alginate lyases hold promise as biochemicals for removing bacterial biofilm alginate which functions as an important virulence factor during lung infections in cystic fibrosis patients. More recently, alginate lyases have been expected to become potential enzymes in the bio-energy generation from alginate. Thus, alginate lyases are important enzymes in a broad spectrum of biological roles and applications. Alginate lyases are classified into7families (family PL5,6,7,14,15,17,18) in the Caborhydrate-Active enZYmes (CAZy) data base. These lyases catalyze the degradation of alginate by a β-elimination of the4-O-glycosyl bond accompanied by the formation of a double bond between C-4and C-5and the production of4-deoxy-L-erythro-hex-4-ene pyranosyluronate at the nonreduceing end of the resulting oligosaccharides. Alginate lyases have been isolated from various sources, namly marine algae, marine mollusks, fungi, bacteria, bacteriophages, and viruses.Various alginate lyases are discoveried from marine bacteria that associated with brown alga. The Arctic Ocean is the most extreme ocean regarding the seasonality of light and its year-round existing ice cover. A large number of unique life forms are highly adapted to the extreme environment of the Arctic Ocean in their ecology and physiology. Brown alga growing in the intertidal zone of the Arctic Ocean is a potential resource for the discovery of new bacteria producing novel alginate lyases, which, however, have never been investigated. In this dissertation, the cultivable alginate lyase-excreting bacteria associated with the Arctic brown alga Laminaria were isolated and the diversity of them were further studied, which constituted a groundwork for mining novle cold-adaped alginate lyases from these Arctic bacterial resources.Up to now, the crystal structures of some alginate lyases have been detrermined. The relationships between structure and function of these enzymes have been well demonstrated. PL18is a relatively new family and mechanisms on the catalysis, substrate recognization and mature of enzymes in this family were therefore not clear. A marine bacterium with high alginate-lyase production, named Psedoalteromonas sp. SM0524, was preiously isolated from Yantai, China in our Lab. Two alginate Iyases are secreted by this bacterium, namely aly SJO1and aly SJ02. aly SJ01is belonged to PL7while aly SJ02, a bifunction alginate lyase with high activities,, is a member of PL18. In this dissertation, the enzymatic properties and mechanisms of substrate specificity for the PL7alginate lyase aly SJO1were firstly studied; crystal structures of family PL18aly SJ02were then determined, through which mechnism of substrate recognition and the function of N-terminal domain are also studied..The results are as following.(1) Isolation and diversity analysis of alginate lyase-extracting bacteria from Arctic Laminaria.A total of74isolates were isolated from6Arctic Laminaria samples and they were further purified and subjected to16S rRNA gene amplification. According to an alignment of the16S rRNA gene sequences, a total of65different strains were isolated from the Arctic Laminaria samples. Phylogenetic analysis based on the16S rRNA gene sequences showed that the isolated Lam/nana-associated bacteria belonged to nine genera. Psychrobacter (33/65) were the most predominant group. Psychromonas (10/65) and Polaribacter (8/65) also showed some preponderance.21algiante lyase-excreting bacteria were further screened from the65 Lammarria-associated bacteria and phylogenetic analysis based on the16S rRNA gene sequences revealed that they were belonged to5genera, namely Pseudoalteromonas, Psychrobacter, Winogradskyella, Psychromonas and Polaribacter. Alginate lyase-excreting marine bacteria so far reported include Alginovibrio, Alteromonas, Beneckea, Halomonas, Photobacterium, Pseudoalteromonas and Vibro. Besides Pseudoalteromonas, it was first found that members of genera Psychrobacter, Winogradskyella, Psychromonas and Polaribacter excreted alginate lyases. Among these Arctic algiante lyase-excreting strains, the optimal temperatures for growth and algiante lyase production of many strains were lower than25℃, showing that they were psychrophilic bacteria. The alginate lyases excreted by11strains showed the highest activity at20-30℃, indicating that these alginate lyases were cold-adapted enzymes. Moreover, some strains showed high extracellular alginate lyase activity levels around200U/mL. These results suggest that the algiante lyase-excreting bacteria from the Arctic alga Laminaria may be good materials for studying bacterial cold-adapted alginate lyases.(2) Enzymatic characterization and substrate specificity mechanism study of the PL7alginate lyase aly SJ01A novel alginate lyase named aly SJ01was cloned from the marine strain Pseudoalteromonas sp. SM0524. Sequence analysis showed that aly SJ01belonged to the PL7family and had only about41%highest identity to a PL7characterized alginate lyase, AlyVGI from Vibrio sp. QY101. aly SJ01was further expressed in Escherichia coli and the enzymatic properties of this enzyme were studied. The optimal temperature of aly Sj01was at around30℃. aly SJ01was rapidly inactivated above30℃and about80%of enzyme activity was lost on preincubation at40℃for15min, indicating the low thermal stability of this lyase.. The apparent melting temperature (Tm) of Aly SJ01determined by Circular Dichroism (CD) was37℃. These results indicated that aly SJ01was a cold-adaptive alginate lyase. The optimal pH of aly SJ01is at8.5; its activity was increased6times by adding0.6M NaCl, a value approximately equal to that in seawater. All the above results suggested that aly SJO1is active and thereby play an important biological role in cold, slightly alkaline and salty marine environments. PolyM, polyG and alginate were respectively reacted wih aly SJO1and the resultant oligosaccharides with different degrees of polymerization were analyzed by TLC. Results from the TLC analysis indicated that enzymatic products were mainly dimmer, trimmer and tetramer with small amounts of monermers and some larger oligosaccharides. The substrate specificity result indicates that aly SJ01had a preference for poly M substrates rather than poly G and poly MG. Despite of the the highest identity with alginate lyase AlyVGI, aly SJ01exhibited different substrate specification from AlyVGI. To better understand the substrate specificity of aly SJ01, amino acids sequence alignments were performed and site-directed mutagenesis were conducted. Analysis focused on mutations in aly SJ01with significant effect on specificity or activity. In summary, conserved amino acids in the active center show little relations to substrate specificity while some introduced mutations in Loop1and Loop2and around the active center apparently increased enzyme activity for polyG with decreased activity towards polyM. Hence, the substrate specificity of aly SJ01seemly results from the unconserved residues around the active center.(3) N-terminal function analysis of PL18alginate lyase aly SJ02The precursor of aly-SJ02contains a signal peptide (Metl-Ala31), an N-terminal domain (Ala32-Gly155)(ND), a linker (Ser156-Ser173) and a PL18catalytic domain (CD)(Thr174-Asn400). Mature aly-SJ02only contains the CD while ND was cut off. The N-terminal domain of aly-SJ02showed alginate binding ability when it was recombinantly expressed individually. However, it showed no binding ability in the precursor of aly SJ02. The enzymatic activity analysis showed that the activity of CD toward alginate was approximately20%lower than that of mature aly SJ02. When the The N-terminal and catalytic domains domain of aly-SJ02were co-expressed with two different vectors in an E. coli cell, the ND and the CD could form a protein complex by interaction, while the folded ND and CD expressed in different E. coli cells could not. To better eluciate that how the ND affects the CD folding, the CD crystal structures from different folding process, namely expressed individually(r-CD) and expressed in precursor(P-CD) were solved. Taking the Psedoalteromonas sp.272alginate lyase (PDB:1J1T) as model, the crystal structure of aly SJ02(M-CD) was obtained by homology modeling. A detail comparison of conformation of these conserved residues in the active centers of r-CD, P-CD and M-CD was preformed. These conserved residues in P-CD and M-CD have the same conformation, suggesting that P-CD has folded correctly. For r-CD, while those residues in molecule B in the crystal unit shares almost the same conformation as those in P-CD (except a tiny swing between Arg219and Tyr353in the two molecules), molecule A differs greatly from P-CD in their conformation. This indicates that, without the presence of the ND, the CD of aly-SJ02may fold disorderly, thereby affecting its activity toward alginate. Base on the above results, it was proposed that the N-terminal domain has evoluted a new function faciliating the folding of catalytic domain.(4) Substrate recognition and catalysis mechanism studies of PL18alginate lyase aly SJ02The PL18alginate lyase aly SJ02had a P-sandwich fold consisting of two β-sheets creating a deep active cleft which was covered by two flex loops. This crystal scaffold was also common shared by the family PL7and PL18alginate lyases. Atomic absorption spectroscopy confirmed that a Ca2+is present in aly-SJ02. The chelate site of Ca2+is far away from the reaction center, suggesting that this metal ion is not directly involved in the polysaccharide degrading reaction. Deprivation of Ca2+from aly-SJ02by EDTA could result in a50%reduce in enzymatic activity. This indicates that the Ca2+in aly-SJ02is important for keeping the enzymatic activity though it does not participate directly in the catalytic reaction. It has been found that alginate lyases usually have loops covering the active cleft. To investigate whether the lid loops of aly-SJ02have a gating function in substrate entry and product release, we performed an Molecular Dynamics simulation for aly-SJ02P-CD structure, with a particular focus on these two loops. Distance measurement of the side chains of Asn214and of Thr263suggests that the space between the loops can increase to11.5A in the "open" state from3.2A in the "closed" state, which makes it possible for an alginate molecule entering the substrate-binding pocket of aly-SJ02. A mutant Asn214Cys/Thr263Cys with Asn214and Thr263being replaced by cysteine residues was constructed to introduce a rigid interaction between loop1and loop2by forming a disulfide bond. This mutation almost completely abolished the activity of aly-SJ02, and a reduction of this disulfide bond by DTT would increase the mutant activity significantly. These result indicates that keeping the flexibility of the lid loops is essential for substrate entry into the substrate-binding pocket of aly-SJ02.Uronic acid residue A-1is accommodated at subsite-1by residues Gln355and Lys364. At subsite+1, the carboxyl group of the saccharide residue A+l is recognized by Gln257and His259, O2by Thr263,03by Asn216, and O4by Thr353. At subsite+2, the carboxyl group is recognized by Arg219and Lys349, and03and O4by His259. At subsite+3, the carboxyl group of A+3is recognized by Lys223and Tyr347. Amino acid substitutions of conserved residues at subsites+1and+2resulted in inactivation of the enzyme while some mutation of those located at subsites-1and+3still reserved enzyme activities. The Km values of the active mutants were all increased, indicating these mutations decreased the affinity of aly-SJ02to the substrate. The residues located at subsites+1and+2are more rigidly conserved than the others, indicating the substrate recognition and binding mainly happened at subsite+1and+2, while residues at the subsites-1and+3have some relatively weak interactions which is consistent with the previous results that aly-SJ02depolymerized alginate and released dimer and trimer mainly.Based on Docking model of the aly-SJ02-oligosaccharides complex and our mutation assays, the catalytic mechanism of aly-SJ02for alginate degradation is proposed. The distance between C5and Tyr353is3.1A, and that between04and Tyr353is3.3A. Mutations of Tyr353resulted in inactivation of the enzyme. Tyr353is function as a catalytic base and acid, which can obtain a proton as a catalytic base from the C5of A+1and give proton to04as a catalytic acid. And residues Arg219, Gln257, His259and Lys349that around subsite+1and+2, stabilizing or neutralizing the negative charge of uronic acid, are quit crucial for substrate recognization and the catalytic reaction,To our knowledge, the above results are the first reports about the substrate recognition and catalytic mechanism of PL18alginate lyases, which will facilitate the studies on structure and function of alginate lyases in this family and provide a foundation for developing new applications of alginate lyases. |
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