William Helbert

Biochemical and Structural Characterization of the Complex Agarolytic Enzyme System from the Marine Bacterium Zobellia Galactanivorans.

Background: Bacterial agarolytic systems are frequent and play an important role in algal biomass conversion. Results: Structural and biochemical analyses of several agar-related enzymes reveal details on substrate recognition and complementary roles. Conclusion: The diversity of agar-related enzymes within a bacterial organism reflects the complexity of the natural substrate. Significance: Marine microbes employ complex systems to catalyze degradation of polysaccharides with unique structural characteristics. Zobellia galactanivorans is an emerging model bacterium for the bioconversion of algal biomass. Notably, this marine Bacteroidetes possesses a complex agarolytic system comprising four β-agarases and five β-porphyranases, all belonging to the glycoside hydrolase family 16. Although β-agarases are specific for the neutral agarobiose moieties, the recently discovered β-porphyranases degrade the sulfated polymers found in various quantities in natural agars. Here, we report the biochemical and structural comparison of five β-porphyranases and β-agarases from Z. galactanivorans. The respective degradation patterns of two β-porphyranases and three β-agarases are analyzed by their action on defined hybrid oligosaccharides. In light of the high resolution crystal structures, the biochemical results allowed a detailed mapping of substrate specificities along the active site groove of the enzymes. Although PorA displays a strict requirement for C6-sulfate in the −2- and +1-binding subsites, PorB tolerates the presence of 3–6-anhydro-l-galactose in subsite −2. Both enzymes do not accept methylation of the galactose unit in the −1 subsite. The β-agarase AgaD requires at least four consecutive agarose units (DP8) and is highly intolerant to modifications, whereas for AgaB oligosaccharides containing C6-sulfate groups at the −4, +1, and +3 positions are still degraded. Together with a transcriptional analysis of the expression of these enzymes, the structural and biochemical results allow proposition of a model scheme for the agarolytic system of Z. galactanivorans.

Enzymatic degradation of hybrid ι-/ν-carrageenan by Alteromonas fortis ι-carrageenase.

Hybrid iota-/nu-carrageenan was water-extracted from Eucheuma denticulatum and incubated with Alteromonas fortis iota-carrageenase. The degradation products were then separated by anion-exchange chromatography. The three most abundant fractions of hybrid iota-/nu-carrageenan oligosaccharides were purified and their structures were analyzed by NMR. The smallest hybrid was an octasaccharide with a iota-iota-nu-iota structure. The second fraction was composed of two decasaccharides with iota-iota-iota-nu-iota and iota-[iota/nu]-iota-iota structures. The third fraction was a mixture of dodecasaccharides which contained at least a iota-iota-iota-iota-nu-iota oligosaccharide. The carbon and proton NMR spectra of the octasaccharides were completely assigned, thereby completely attributing the nu-carrabiose moiety for the first time.

Physical state of κ-carrageenan modulates the mode of action of κ-carrageenase from Pseudoalteromonas carrageenovora

Pseudoalteromonas carrageenovora kappa-carrageenase is a glycoside hydrolase involved in the bioconversion of carrageenans. Carrageenans are sulfated galactans that are densely packed in red algal cell walls. Previous crystallographic investigations revealed that the active site of kappa-carrageenase has a tunnel-shaped topology, suggesting a processive mode of action for this enzyme. To biochemically characterize the enzymatic depolymerization of kappa-carrageenan, soluble and solid substrates (in both gel and powder forms) were incubated with P. carrageenovora kappa-carrageenase. The average molecular mass of soluble carrageenan decreased rapidly, and all possible degradation products were observed, suggesting random degradation of kappa-carrageenan. In contrast, as expected for a processive-type carrageenase, the average molecular mass of solid carrageenan decreased very slowly, and tetrasaccharide production was high. Interestingly, experimentally determined processivity was similar for gel and powder, suggesting that, in addition to an adapted catalytic site, the substrate must be in the solid state for kappa-carrageenase processivity to operate, whatever the level of carrageenan ordering.

A Novel Unsaturated β-Glucuronyl Hydrolase Involved in Ulvan Degradation Unveils the Versatility of Stereochemistry Requirements in Family GH105

Background: Biodegradation of green algal cell wall requires specialized enzymatic machinery, which is not yet well characterized. Results: Structural and biochemical characterization of a new β-glucuronyl hydrolase belonging to family GH105 active on oligo-ulvans. Conclusion: The GH105 family encompasses enzymes cleaving both α- and β-linked glycosides. Significance: Investigations of enzymatic degradation of marine polysaccharides reveals enzymes with unique characteristics. Ulvans are cell wall matrix polysaccharides in green algae belonging to the genus Ulva. Enzymatic degradation of the polysaccharide by ulvan lyases leads to the production of oligosaccharides with an unsaturated β-glucuronyl residue located at the non-reducing end. Exploration of the genomic environment around the Nonlabens ulvanivorans (previously Percicivirga ulvanivorans) ulvan lyase revealed a gene highly similar to known unsaturated uronyl hydrolases classified in the CAZy glycoside hydrolase family 105. The gene was cloned, the protein was overexpressed in Escherichia coli, and enzymology experiments demonstrated its unsaturated β-glucuronyl activity. Kinetic analysis of purified oligo-ulvans incubated with the new enzyme showed that the full substrate specificity is attained by three subsites that preferentially bind anionic residues (sulfated rhamnose, glucuronic/iduronic acid). The three-dimensional crystal structure of the native enzyme reveals that a trimeric organization is required for substrate binding and recognition at the +2 binding subsite. This novel unsaturated β-glucuronyl hydrolase is part of a previously uncharacterized subgroup of GH105 members and exhibits only a very limited sequence similarity to known unsaturated β-glucuronyl sequences previously found only in family GH88. Clan-O formed by families GH88 and GH105 was singular in the fact that it covered families acting on both axial and equatorial glycosidic linkages, respectively. The overall comparison of active site structures between enzymes from these two families highlights how that within family GH105, and unlike for classical glycoside hydrolysis, the hydrolysis of vinyl ether groups from unsaturated saccharides occurs independently of the α or β configuration of the cleaved linkage.

Controlling Carrageenan Structure Using a Novel Formylglycine-Dependent Sulfatase, an Endo-4S-iota-Carrageenan Sulfatase

Carrageenans are sulfated polysaccharides that are found in the cell walls of red algae. These polysaccharides have gelling and texturizing properties that are widely appreciated in industrial applications. However, these functional properties depend strongly on the sulfation of the moieties of the carrabiose repetition unit. Here we aimed to monitor the sulfate composition of gelling carrageenan. To do so, we screened and purified from Pseudoalteromonas atlantica a 4S-iota carrageenan sulfatase that converts ι-carrabiose into α-carrabiose units. The sequence of this protein matched the annotated Q15XH3 (Uniprot databank) formylglycine-dependent sulfatase found in the P. atlantica genome. With pure enzyme, ι-carrageenan could be transformed into a hybrid ι-/α-carrageenan or pure α-carrageenan. Analysis of the distribution of the carrabiose moieties in hybrid carrageenan chain using enzymatic degradation with Alteromonas fortis ι-carrageenase, coupled with chromatography and NMR spectroscopy experiments, showed that the sulfatase has an endo mode of action. The endo-character and the specificity of the sulfatase made it possible to prepare hybrid κ-/ι-/α-carrageenan and κ-/α-carrageenan starting from κ-/ι-carrageenan.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Polysulfated-Derived Oligosaccharides Using Pyrenemethylguanidine

A better understanding of the biological roles of carbohydrates requires the use of tools able to provide efficient and rapid structural information. Unfortunately, highly acidic oligomers—such as polysulfated oligosaccharides—are very challenging to characterize because of their high polarity, structural diversity, and sulfate lability. These features pose special problems for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis because polysulfated carbohydrates exhibit poor ionization efficiency and usually do not produce any signal. The present report demonstrates how MALDI-MS can be used to derive structural and compositional information from pure and mixed fractions of polysulfated oligosaccharides. Indeed, pyrenemethylguanidine (pmg, a derivatizing agent and ionization efficiency enhancer) was used for the analysis of di- to decasaccharides, carrying from two to nine sulfate groups. The method is applied to various highly sulfated chondroitin and carrageenan oligosaccharides as well as to the analysis of mixtures of compounds. In the mass spectra, the observation of a unique pmg-complexed ladder of peaks in both ionization modes allows an easy and rapid determination of both the number of sulfate groups carried by the analyte and its molecular weight. Moreover, we have developed a software tool for the rapid and automatic structural elucidation of carrageenans based on the mass spectra obtained.

Complete assignment of 1H and 13C NMR spectra of standard neo-ι-carrabiose oligosaccharides.

Standard Eucheuma denticulatum iota-carrageenan was degraded with the Alteromonas fortis iota-carrageenase. The most abundant products, the neo-iota-carratetraose and neo-iota-carrahexaose were purified by permeation gel chromatography, and their corresponding (1)H and (13)C NMR spectra were fully assigned.

Hybridity of carrageenans water- and alkali-extracted from Chondracanthus chamissoi, Mazzaella laminarioides, Sarcothalia crispata, and Sarcothalia radula

Carrageenans water- and alkali-extracted from Chondracanthus chamissoi, Mazzaella laminarioides, Sarcothalia crispata, and Sarcothalia radula were degraded by Pseudoalteromonas carrageenovora κ-carrageenase and Alteromonas fortis ι-carrageenase. The composition of the high molecular weight fraction (i.e., enzyme-resistant fraction) as well as the standard and hybrid oligosaccharide content highlighted differences between the species-specific carrageenans in terms of composition and distribution of the carrabiose moieties. Inspection of the distribution of μ- and ν-carrabiose, the biosynthetic precursors of κ- and ι-carrabiose, revealed their localization in κ-carrabiose- or ι-carrabiose-rich segments, respectively.