ι-Carrageenan catabolism is initiated by key sulfatases in the marine bacterium Pseudoalteromonas haloplanktis LL1.

Marine bacteria contribute substantially to cycle macroalgae polysaccharides in marine environments. Carrageenans are the primary cell wall polysaccharides of red macroalgae. The carrageenan catabolism mechanism and pathways are still largely unclear. Pseudoalteromonas is a representative bacterial genus that can utilize carrageenan. We previously isolated the strain Pseudoalteromonas haloplanktis LL1 that could grow on ι-carrageenan but produce no ι-carrageenase. Here, through a combination of bioinformatic, biochemical, and genetic analyses, we determined that P. haloplanktis LL1 processed a desulfurization-depolymerization sequential pathway for ι-carrageenan utilization, which was initiated by key sulfatases PhSulf1 and PhSulf2. PhSulf2 acted as an endo/exo-G4S (4-O-sulfation-ß-D-galactopyranose) sulfatase, while PhSulf1 was identified as a novel endo-DA2S sulfatase that could function extracellularly. Because of the unique activity of PhSulf1 toward ι-carrageenan rather than oligosaccharides, P. haloplanktis LL1 was considered to have a distinct ι-carrageenan catabolic pathway compared to other known ι-carrageenan-degrading bacteria, which mainly employ multifunctional G4S sulfatases and exo-DA2S (2-O-sulfation-3,6-anhydro-α-D-galactopyranose) sulfatase for sulfate removal. Furthermore, we detected widespread occurrence of PhSulf1-encoding gene homologs in the global ocean, indicating the prevalence of such endo-acting DA2S sulfatases as well as the related ι-carrageenan catabolism pathway. This research provides valuable insights into the enzymatic processes involved in carrageenan catabolism within marine ecological systems.IMPORTANCECarrageenan is a type of linear sulfated polysaccharide that plays a significant role in forming cell walls of marine algae and is found extensively distributed throughout the world's oceans. To the best of our current knowledge, the ι-carrageenan catabolism in marine bacteria either follows the depolymerization-desulfurization sequential process initiated by ι-carrageenase or starts from the desulfurization step catalyzed by exo-acting sulfatases. In this study, we found that the marine bacterium Pseudoalteromonas haloplanktis LL1 processes a distinct pathway for ι-carrageenan catabolism employing a specific endo-acting DA2S-sulfatase PhSulf1 and a multifunctional G4S sulfatase PhSulf2. The unique PhSulf1 homologs appear to be widely present on a global scale, indicating the indispensable contribution of the marine bacteria containing the distinct ι-carrageenan catabolism pathway. Therefore, this study would significantly enrich our understanding of the molecular mechanisms underlying carrageenan utilization, providing valuable insights into the intricate roles of marine bacteria in polysaccharide cycling in marine environments.

The structure of a pectin-active family 1 polysaccharide lyase from the marine bacterium Pseudoalteromonas fuliginea.

Pseudoalteromonas fuliginea sp. PS47 is a recently identified marine bacterium that has extensive enzymatic machinery to metabolize polysaccharides, including a locus that targets pectin-like substrates. This locus contains a gene (locus tag EU509_03255) that encodes a pectin-degrading lyase, called PfPL1, that belongs to polysaccharide lyase family 1 (PL1). The 2.2 Šresolution X-ray crystal structure of PfPL1 reveals the compact parallel ß-helix fold of the PL1 family. The back side of the core parallel ß-helix opposite to the active site is a meandering set of five α-helices joined by lengthy loops. A comparison of the active site with those of other PL1 enzymes suggests a catalytic mechanism that is independent of metal ions, such as Ca2+, but that substrate recognition may require metal ions. Overall, this work provides the first structural insight into a pectinase of marine origin and the first structure of a PL1 enzyme in subfamily 2.

Improvement of thermostability by increasing rigidity in the finger regions and flexibility in the catalytic pocket area of Pseudoalteromonas porphyrae κ-carrageenase.

Poor thermostability reduces the industrial application value of κ-carrageenase. In this study, the PoPMuSiC algorithm combined with site-directed mutagenesis was applied to improve the thermostability of the alkaline κ-carrageenase from Pseudoalteromonas porphyrae. The mutant E154A with improved thermal stability was successfully obtained using this strategy after screening seven rationally designed mutants. Compared with the wild-type κ-carrageenase (WT), E154A improved the activity by 29.4% and the residual activity by 51.6% after treatment at 50 °C for 30 min. The melting temperature (Tm) values determined by circular dichroism were 66.4 °C and 64.6 °C for E154A and WT, respectively. Molecular dynamics simulation analysis of κ-carrageenase showed that the flexibility decreased within the finger regions (including F1, F2, F3, F5 and F6) and the flexibility improved in the catalytic pocket area of the mutant E154A. The catalytic tunnel dynamic simulation analysis revealed that E154A led to enlarged catalytic tunnel volume and increased rigidity of the enzyme-substrate complex. The increasing rigidity within the finger regions and more flexible catalytic pocket of P. porphyrae κ-carrageenase might be a significant factor for improvement of the thermostability of the mutant κ-carrageenase E154A. The proposed rational design strategy could be applied to improve the enzyme kinetic stability of other industrial enzymes. Moreover, the hydrolysates of κ-carrageenan digested by the mutant E154A demonstrated increased scavenging activities against hydroxyl (OH) radicals and 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radicals compared with the undigested κ-carrageenan.

A Novel Alginate Lyase: Identification, Characterization, and Potential Application in Alginate Trisaccharide Preparation.

Alginate oligosaccharides (AOS) have many biological activities and significant applications in prebiotics, nutritional supplements, and plant growth development. Alginate lyases have unique advantages in the preparation of AOS. However, only a limited number of alginate lyases have been so far reported to have potentials in the preparation of AOS with specific degrees of polymerization. Here, an alginate-degrading strain Pseudoalteromonasarctica M9 was isolated from Sargassum, and five alginate lyases were predicted in its genome. These putative alginate lyases were expressed and their degradation products towards sodium alginate were analyzed. Among them, AlyM2 mainly generated trisaccharides, which accounted for 79.9% in the products. AlyM2 is a PL6 lyase with low sequence identity (≤28.3%) to the characterized alginate lyases and may adopt a distinct catalytic mechanism from the other PL6 alginate lyases based on sequence alignment. AlyM2 is a bifunctional endotype lyase, exhibiting the highest activity at 30 °C, pH 8.0, and 0.5 M NaCl. AlyM2 predominantly produces trisaccharides from homopolymeric M block (PM), homopolymeric G block (PG), or sodium alginate, with a trisaccharide production of 588.4 mg/g from sodium alginate, indicating its promising potential in preparing trisaccharides from these polysaccharides.

Identification and Characterization of Three Chitinases with Potential in Direct Conversion of Crystalline Chitin into N,N'-diacetylchitobiose.

Chitooligosaccharides (COSs) have been widely used in agriculture, medicine, cosmetics, and foods, which are commonly prepared from chitin with chitinases. So far, while most COSs are prepared from colloidal chitin, chitinases used in preparing COSs directly from natural crystalline chitin are less reported. Here, we characterize three chitinases, which were identified from the marine bacterium Pseudoalteromonas flavipulchra DSM 14401T, with an ability to degrade crystalline chitin into (GlcNAc)2 (N,N'-diacetylchitobiose). Strain DSM 14401 can degrade the crystalline α-chitin in the medium to provide nutrients for growth. Genome and secretome analyses indicate that this strain secretes six chitinolytic enzymes, among which chitinases Chia4287, Chib0431, and Chib0434 have higher abundance than the others, suggesting their importance in crystalline α-chitin degradation. These three chitinases were heterologously expressed, purified, and characterized. They are all active on crystalline α-chitin, with temperature optima of 45-50 °C and pH optima of 7.0-7.5. They are all stable at 40 °C and in the pH range of 5.0-11.0. Moreover, they all have excellent salt tolerance, retaining more than 92% activity after incubation in 5 M NaCl for 10 h at 4 °C. When acting on crystalline α-chitin, the main products of the three chitinases are all (GlcNAc)2, which suggests that chitinases Chia4287, Chib0431, and Chib0434 likely have potential in direct conversion of crystalline chitin into (GlcNAc)2.

Genomic and in silico protein structural analyses provide insights into marine polysaccharide-degrading enzymes in the sponge-derived Pseudoalteromonas sp. PA2MD11.

Active heterotrophic metabolism is a critical metabolic role performed by sponge-associated microorganisms, but little is known about their capacity to metabolize marine polysaccharides (MPs). Here, we investigated the genome of the sponge-derived Pseudoalteromonas sp. strain PA2MD11 focusing on its macroalgal carbohydrate-degrading potential. Carbohydrate-active enzymes (CAZymes) for the depolymerization of agar and alginate were found in PA2MD11's genome, including glycoside hydrolases (GHs) and polysaccharide lyases (PLs) belonging to families GH16, GH50 and GH117, and PL6 and PL17, respectively. A gene potentially encoding a sulfatase was also identified, which may play a role in the strain's ability to consume carrageenans. The complete metabolism of agar and alginate by PA2MD11 could also be predicted and was consistent with the results obtained in physiological assays. The polysaccharide utilization locus (PUL) potentially involved in the metabolism of agarose contained mobile genetic elements from other marine Gammaproteobacteria and its unusual larger size might be due to gene duplication events. Homology modelling and structural protein analyses of the agarases, alginate lyases and sulfatase depicted clear conservation of catalytic machinery and protein folding together with suitable industrially-relevant features. Pseudoalteromonas sp. PA2MD11 is therefore a source of potential MP-degrading biocatalysts for biorefinery applications and in the preparation of pharmacologically-active oligosaccharides.

Degradation and Utilization of Alginate by Marine Pseudoalteromonas: a Review.

Alginate, which is mainly produced by brown algae and decomposed by heterotrophic bacteria, is an important marine organic carbon source. The genus Pseudoalteromonas contains diverse forms of heterotrophic bacteria that are widely distributed in marine environments and are an important group in alginate degradation. In this review, the diversity of alginate-degrading Pseudoalteromonas is introduced, and the characteristics of Pseudoalteromonas alginate lyases, including their sequences, enzymatic properties, structures, and catalytic mechanisms, and the synergistic effect of Pseudoalteromonas alginate lyases on alginate degradation are introduced. The acquisition of the alginate degradation capacity and the alginate utilization pathways of Pseudoalteromonas are also introduced. This paper provides a comprehensive overview of alginate degradation by Pseudoalteromonas, which will contribute to the understanding of the degradation and recycling of marine algal polysaccharides driven by marine bacteria.

Module function analysis of a full-length κ-carrageenase from Pseudoalteromonas sp. ZDY3.

κ-Carrageenan oligosaccharides with many excellent biological properties could be produced by κ-carrageenases selectively. In this study, based on the encoding gene of full length κ-carrageenase obtained from Pseudoalteromonas sp. ZDY3 and the reported mature secreted κ-carrageenase composed of 275 amino acid residues (N26-T300), CgkPZ_GH16 was expressed in E. coli, but no soluble active protein could be detected. Fortunately, the signal peptide of wild-type κ-carrageenase was recognized, and cleaved in the soluble and folding form in E. coli, the Km and kcat values of CgkPZ_SP_GH16 was 1.007 mg/mL and 362.8 s-1. By molecular dynamics simulations, it was showed that YjdB domain might affect the activity of κ-carrageenase. Due to the absence of mature processing modification system in E. coli, YjdB was remained in recombinant full length κ-carrageenase, and the lost catalytic efficiency of CgkPZ was compensated by expression level and thermal stability. Interestingly, CgkPZ_GH16_YjdB was expressed soluble without the signal peptide, which indicated that YjdB could contribute to the expression and folding of κ-carrageenase. These results provide new insight into the effects of different modules of κ-carrageenase on the expression and properties of enzyme.

Purification and characterization of a cold-adapted κ-carrageenase from Pseudoalteromonas sp. ZDY3.

κ-Carrageenase (EC3.2.1.83) is a class of glycoside hydrolase, which can be used for hydrolysis of κ-carrageenan to κ-carrageenan oligosaccharides. In this study, a bacterium, identified as Pseudoalteromonas sp. ZDY3 isolated from rotten algae, was capable to degrade κ-carrageenan. The κ-carrageenase produced by Pseudoalteromonas sp. ZDY3 was purified to homogeneity and named as CgkZDY3. The accurate molecular mass of CgkZDY3 was determined through LC-HRMS, and a posttranslational removal of C-terminal end of the protein was discovered. CgkZDY3 had strict hydrolysis specificity to κ-carrageenan, the values of Km and kcat/Km of CgkZDY3 were 3.67 mg mL-1 and 53.0 mL mg-1 s-1, respectively. CgkZDY3 was a cold-adapted κ-carrageenase with excellent storage stability of both the temperature below 35 °C and a wide pH range, and was an endo-type κ-carrageenase with high hydrolysis rate, oligosaccharides with different degrees of polymerization can be obtained by controlling the hydrolysis time, and the final products were κ-neocarrabiose and κ-neocarratetraose. These properties are of great significance for production of κ-carrageenan oligosaccharides with different polymerization degrees under process control.

The structure of a family 110 glycoside hydrolase provides insight into the hydrolysis of α-1,3-galactosidic linkages in λ-carrageenan and blood group antigens.

α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1-3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure-function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1-3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1-3Gal revealed the parallel ß-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.

A highly divergent α-amylase from Streptomyces spp.: An evolutionary perspective.

The present study deals with the genetic changes observed in the protein sequence of an α-amylase from Streptomyces spp. and its structural homologs from Pseudoalteromonas haloplanktis, invertebrates and mammals. The structural homologs are renowned for their important features such as chloride binding triad and a serine-protease like catalytic triad (a triad which is reported to be strictly conserved in all chloride-dependent α-amylases). These conserved regions are essential for allosteric activation of enzyme and conformational stability, respectively. An evaluation of these distinctive features in Streptomyces α-amylases revealed the role of mutations in conserved regions and evolution of chloride-independent α-amylases in Streptomyces spp. Besides, the study also discovers a highly divergent α-amylase from Streptomyces spp. which varies greatly even within the homologs of the same genus. Another very important feature is the number of disulfide bridges in which the structural homologs own eight Cys residues to form four disulfide bridges whereas Streptomyces α-amylases possess only seven Cys to form three disulfide bridges. The study also highlights the unique evolution of carbohydrate binding module 20 domain (CBM20 also known as raw starch binding domain or E domain) in Streptomyces α-amylases which is completely absent in α-amylases of other structural homologs.

Hidden Conformational States and Strange Temperature Optima in Enzyme Catalysis.

The existence of temperature optima in enzyme catalysis that occur before protein melting sets in can be described by different types of kinetic models. Such optima cause distinctly curved Arrhenius plots and have, for example, been observed in several cold-adapted enzymes from psychrophilic species. The two main explanations proposed for this behavior either invoke conformational equilibria with inactive substrate-bound states or postulate differences in heat capacity between the reactant and transition states. Herein, we analyze the implications of the different types of kinetic models in terms of apparent activation enthalpies, entropies, and heat capacities, using the catalytic reaction of a cold-adapted α-amylase as a prototypic example. We show that the behavior of these thermodynamic activation parameters is fundamentally different between equilibrium and heat capacity models, and in the α-amylase case, computer simulations have shown the former model to be correct. A few other enzyme-catalyzed reactions are also discussed in this context.

The structure of PfGH50B, an agarase from the marine bacterium Pseudoalteromonas fuliginea PS47.

The recently identified marine bacterium Pseudoalteromonas fuliginea sp. PS47 possesses a polysaccharide-utilization locus dedicated to agarose degradation. In particular, it contains a gene (locus tag EU509_06755) encoding a ß-agarase that belongs to glycoside hydrolase family 50 (GH50), PfGH50B. The 2.0 Šresolution X-ray crystal structure of PfGH50B reveals a rare complex multidomain fold that was found in two of the three previously determined GH50 structures. The structure comprises an N-terminal domain with a carbohydrate-binding module (CBM)-like fold fused to a C-terminal domain by a rigid linker. The CBM-like domain appears to function by extending the catalytic groove of the enzyme. Furthermore, the PfGH50B structure highlights key structural features in the mobile loops that may function to restrict the degree of polymerization of the neoagaro-oligosaccharide products and the enzyme processivity.

Characterization of a GH3 halophilic ß-glucosidase from Pseudoalteromonas and its NaCl-induced activity toward isoflavones.

A novel ß-glucosidase gene was isolated from Pseudoalteromonas sp. GXQ-1 and heterologously expressed in Escherichia coli. The activity of the encoded enzyme, PABGL, toward p-nitrophenyl-ß-D-glucopyranoside was increased 8.74-fold by the presence of 3 M NaCl relative to the absence of added NaCl. PABGL hydrolyzed a variety of soy isoflavone substrates. For the conversion of daidzin to daidzein, the production rate was 1.44 mM/h. The addition of NaCl enhanced the hydrolytic activity of PABGL toward daidzin and genistein; the maximum activation by NaCl was 3.48- and 6.79-fold, respectively. This is the first report of a halophilic ß-glucosidase from Pseudoalteromonas spp., and represents the ß-glucosidase with the highest multiple of activation by NaCl. PABGL exhibits strong potential for applications in food processing and industrial production.

Improving the thermostability of a GH97 dextran glucosidase by rational design.

This study was aimed at improving the thermostability of dextran glucosidase PspAG97A, a member of the glycoside hydrolase family 97, from Pseudoalteromonas sp. K8. A total of 9 lysine residues were chosen using the TKSA-MC program based on the optimization of surface charge-charge interactions and were mutated to glutamate for shifting the enzyme's isoelectric point off its optimum pH value. Three mutants K75E, K363E and K420E showed enhanced thermostability. The triple mutant, K75E/K363E/K420E, was found to be the best with a 7.3-fold increase in half-life (t1/2) at 33 °C compared to that of the wild-type (WT). Most importantly, this mutant showed comparable enzymatic activity to that of the WT protein. Structural modelling demonstrated that increased surface charge-charge interactions and optimization of surface hydrophobic and electrostatic contacts contributed to the improved thermostability displayed by K75E/K363E/K420E.

Computer simulations explain the anomalous temperature optimum in a cold-adapted enzyme.

Cold-adapted enzymes from psychrophilic species show the general characteristics of being more heat labile, and having a different balance between enthalpic and entropic contributions to free energy barrier of the catalyzed reaction compared to mesophilic orthologs. Among cold-adapted enzymes, there are also examples that show an enigmatic inactivation at higher temperatures before unfolding of the protein occurs. Here, we analyze these phenomena by extensive computer simulations of the catalytic reactions of psychrophilic and mesophilic α-amylases. The calculations yield temperature dependent reaction rates in good agreement with experiment, and also elicit the anomalous rate optimum for the cold-adapted enzyme, which occurs about 15 °C below the melting point. This result allows us to examine the structural basis of thermal inactivation, which turns out to be caused by breaking of a specific enzyme-substrate interaction. This type of behaviour is also likely to be relevant for other enzymes displaying such anomalous temperature optima.

Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase.

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

A mutant of Pseudoalteromonas carrageenovora arylsulfatase with enhanced enzyme activity and its potential application in improvement of the agar quality.

Enzymatic desulfation using arylsulfatase provides an attractive approach to improve agar quality. We have previously characterized a functional arylsulfatase from Pseudoalteromonas carrageenovora. To further improve its enzymatic performance, we isolated a mutant arylsulfatase of K253Q with improved enzyme activity from a random mutant library. Compared to wild-type arylsulfatase (WT), K253Q showed 33% increase in enzyme activity, with optimal temperature and pH of 55 °C and 8.0, respectively. K253Q demonstrated better substrate binding ability with lower Km value. Structure analysis indicated that a combination of the additional hydrogen bond and the enhanced substrate binding affinity could account for the improved enzyme activity of K253Q. K253Q exhibited about 54% sulfate removal against agar, resulting in additional 8% increase in 3,6-AG content and 20% increase in gel strength compared to WT. Scanning electron microscopy showed that K253Q treatment led to a stronger crosslinking structure of agar.

Enzymatic preparation of chitooligosaccharides and their anti-obesity application.

Chitooligosaccharides (COS) are derived from chitosan, which can be used as nutraceuticals and functional foods. Because of their various biological activities, COS are widely used in the food, medicine, agriculture, and other fields. COS were prepared by chitosanase  from Pseudoalteromonas sp. SY39 and their anti-obesity activity was researched in mice in this study. The effects of hydrolysis time, temperature, the ratio of enzyme to chitosan, and pH on the productivity of COS were discussed. Preparation process of COS was established in a 5-L fermenter. COS were characterized and their anti-obesity activity was studied in animal experiments. The results showed that COS could effectively reduce serum lipid levels and obesity in mice, and have a good anti-obesity activity. The preparation technology and remarkable anti-obesity activity of COS further expand their applications in the food and pharmaceutical industries.

Characterization and Application of an Alginate Lyase, Aly1281 from Marine Bacterium Pseudoalteromonas carrageenovora ASY5.

Alginate extracted from widely cultured brown seaweed can be hydrolyzed by alginate lyase to produce alginate oligosaccharides (AOS) with intriguing biological activities. Herein, a novel alginate lyase Aly1281 was cloned from marine bacterium Pseudoalteromonas carrageenovora ASY5 isolated from mangrove soil and found to belong to polysaccharide lyase family 7. Aly1281 exhibited maximum activity at pH 8.0 and 50 °C and have broad substrate specificity for polyguluronate and polymannuronate. Compared with other alginate lyases, Aly1281 exhibited high degradation specificity and mainly produced di-alginate oligosaccharides which displayed good antioxidant function to reduce ferric and scavenge radicals such as hydroxyl, ABTS+ and DPPH. Moreover, the catalytic activity and kinetic performance of Aly1281 were highly improved with the addition of salt, demonstrating a salt-activation property. A putative conformational structural feature of Aly1281 was found by MD simulation analysis for understanding the salt-activation effect.