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Subsites in glycoside hydrolases
Within the substrate binding cleft of an enzyme, positive (+) and negative (-) substrate binding subsites can be defined, with respect to the catalytic residues, at the point of cleavage. This nomenclature has been adopted for all glycoside hydrolases, whereby definition, –n subsites represent enzyme/sugar ring interactions with the glycon (new reducing end) and +n subsites represent the interactions with the aglycon (new non-reducing end) (Davies et al. 1997). By definition the point of glycosidic bond cleavage is situated between the -1 and +1 binding sites (Figure 14).
Agarolytic bacteria and glycoside hydrolases
The first agar-degrading microorganisms were isolated from a Norwegian fiord by Gran in 1902. Since then at least 30 microorganisms with agarolytic activity have been reported. The vast majority of these bacteria are of marine origin belonging to Cytophaga (Duckworth et al. 1968; Duckworth et al. 1969; Duckworth et al. 1969; Van der Meulen et al. 1975), Microbulbifer (Ohta et al. 2004), Pseudomonas (Ha et al. 1997; Kang et al. 2003), Pseudoalteromonas (Belas 1989; Vera et al. 1998; Ivanova et al. 2003; Schroeder et al. 2003), Microscilla (Zhong et al. 2001), Vibrio (Aoki et al. 1990; Sugano et al. 1993; Sugano et al. 1994; Sugano et al. 1994; Araki et al. 1998), Alterococcus (Shieh et al. 1998), Alteromonas (Potin et al. 1993), Thalassomonas (Ohta et al. 2005) , Saccharophagus (Ekborg et al. 2005; Ekborg et al. 2006) and Zobellia (Allouch et al. 2003; Jam et al. 2005). Agarase activity has also been observed in terrestrial organisms, such as Paenibacillus (Hosoda et al. 2003) and Streptomyces (Bibb et al. 1987); genes of agarases have been found in soil by metagenomics (Voget et al. 2003), and interestingly agarase activity was found in an unidentified hospital contaminant (Swartz et al. 1959). Currently there is only one report of an agarase purified from an eukaryote, the mussel Littorina mandshurica (Usov et al. 1975). Because agarolytic bacteria have been isolated from various far eastern mussels, it is likely that the agarase activity reported for L. mandshurica results from an associated bacterial symbiont.
The glycoside hydrolase family GH16
As mentioned previously, the family classification of glycoside hydrolases is sequence based. A major consequence of this is that the catalytic mechanism is in general the same throughout a GH-family of enzymes (Davies et al. 1995; Henrissat et al. 1997). However, recent exceptions have been reported (Gloster et al. 2008). Another consequence is that while some families contain enzymes displaying one and unique specificity, as family GH6 for cellulases or GH11 for xylanases, others including GH1, GH5 or GH13 covers a wide range of substrate specificities. The glycoside hydrolase family 16 (GH16) is such a polyspecific family with eight described specificities and includes more than 900 sequences to date. Numerous structures have been described, two of which are active on red algal galactans, namely the β-agarases and a κ- carrageenase. All GH16 enzymes contain the same catalytic residues and share the retaining reaction mechanism with retention of the configuration at the anomeric carbon (Keitel et al. 1993). The eight enzyme activities that have been described so far cover lichenases, xyloglucan endotransferases, keratan-sulfate endo-1,4-beta-galactosidases, glucan endo-1,3-beta-D glucosidases, endo-1,3(4)-beta-glucanases, xyloglucanases, β-agarases and κ-carrageenases to which we can add the new activity of β-porphyranases described in my thesis work (Table 2).
The β-agarases from Z. galactanivorans
The first GH16 enzyme purified from Z. galactanivorans was a κ-carrageenase which was expressed by the bacterium in presence of carrageenan as substrate. This enzyme could be purified to homogeneity from the culture supernatant and was subsequently cloned and characterized (Potin et al. 1991; Barbeyron et al. 1998). When Z. galactanivorans is cultivated in the presence of agar the culture supernatant contains β-agarase activity specific for the β-1,4 glycosidic linkages in the polysaccharide. At least two agarase genes coding for different β- agarases were identified by functional cloning in Z. galactanivorans (Jam et al. 2005). These genes were named agaA and agaB. The product of gene agaA codes for a protein with 539 amino acids and contains a N-terminal sequence of 19 residues which probably targets the gene product into the extracellular medium (von Heijne 1983) and is cleaved of in the mature protein (Figure 16). Further sequence analysis revealed that the gene product of agaA is modular, and contains three domains which are the catalytic GH16 module coupled to two C-terminal modules of unknown function. The high affinity of the native AgaA enzyme to sepharose beads (cross linked agarose) suggests that these non characterized modules might consist of agarose specific carbohydrate binding modules (Murielle Jam personal communication).
The “knowledge gap” of marine polysaccharides degrading enzymes
In the past, advances in microbiology, including marine microbiology, depended mostly upon culturing. The new age of metagenomics enables the study of the vast majority of microbial species which are as yet unable to be cultivated in the laboratory. These technologies and the analyses they enable (comparative (meta)genomics, (meta)transcriptomics, (meta)proteomics, metabolomics, high throughput gene disruptions, etc) have ushered in a new era of biology with fundamental implications for basic research and biotechnological advances. But, they also pose challenges, especially in marine biology, since this flow of data also highlight the lack of knowledge concerning marine specific metabolisms.
Table of contents :
Summary
I. Introduction
I.1 The marine ecosystem
I.1.1 Heterotrophic bacteria as key player in marine carbon cycle
I.1.2 Polysaccharides are an important part of the marine DOM and POM pools
The « Sweet Ocean »
I.1.3 Bacterial enzymatic decomposition of marine organic matter
I.1.4 Why enzymatic degradation of gel forming marine galactans impacts the carbon cycle
The ocean is a « Sweet Jelly »
I.2 Marine polysaccharides
I.2.1 The cell walls of marine macrophytes
I.2.2 The skeleton component of algal cell walls
I.2.3 The matrix component of algal cell walls
I.2.4 The green algae
I.2.5 The brown algae
I.2.6 The cell wall matrix of marine red algae: agars and carrageenans
I.2.7 Agarose 3D structure
I.3 The marine heterotrophic bacteria
I.3.1 Marine bacteroidetes
I.3.2 Zobellia galactanivorans
I.4 The glycoside hydrolases
I.4.1 Enzymatic transformation of HMW compounds
I.4.2 Glycoside hydrolases and their sequence based classification
I.4.3 The catalytic mechanism of glycoside hydrolases
I.4.4 Mode of action in glycoside hydrolases
I.4.5 Subsites in glycoside hydrolases
I.4.6 Agarolytic bacteria and glycoside hydrolases
I.4.7 Family classification of agarases
I.4.8 The glycoside hydrolase family GH16
I.4.9 The β-agarases from Z. galactanivorans
I.5 The “knowledge gap” of marine polysaccharides degrading enzymes
I.6 The aim of the thesis: finding new glycoside hydrolases by analysing the agarolytic system of Zobellia galactanivorans
Structural and Functional Organisation of the Agarolytic Enzyme System of the Marine Flavobacterium Zobellia galactanivorans
II. Results
II.1 Medium throughput cloning and expression strategy
II.2 The targets for further characterization: AgaD, PorA and PorB
III. The Structural and Biochemical Characterization of the new -agarase Agad
III.1 Introduction for manuscript1: Protein crystallization of AgaD
III.2 Manuscript 1
III.2.1 Abstract
III.2.2 Introduction
III.2.3 Material and Methods
III.2.4 Results and discussion
III.2.5 Conclusion
III.2.6 Acknowledgments
III.2.7 References
III.3 The crystal structure of AgaD
III.4 Biochemical characterization of AgaD
III.4.1 Catalytic behaviour of AgaD
III.4.2 AgaD is an endo β-agarase cleaving the β-1,4 linkages in agarose
III.4.3 Agarase specificities are different on natural substrates extracted from the agarophytes Gelidium, and Porphyra
III.4.4 PACE and HPLC analysis of porphyran degradation by AgaA, AgaB and AgaD .97
III.5 Conclusion: The new -agarase AgaD together with AgaA,B as part of the agarolytic enzyme system of Z. galactanivorans
IV. The first -porphyranases PorA and PorB
IV.1 Crystallisation of PorA
IV.1.1 3D structure solution of PorA using a gold derivative
IV.1.2 Crystallization of PorB
IV.1.3 The crystal structures of PorA and PorB
IV.2 The discovery of the β-porphyranase activity
IV.3 Introduction for manuscript2: Porphyranases and agarases constitute the first example of a nutrition derived CAZyme update into human gut bacteria
IV.4 Manuscript 2
IV.4.1 Abstract
IV.4.2 Discovering a new enzyme activity
IV.4.3 Structural determinants of porphyran active enzymes
IV.4.4 β-Porphyranases are abundant in marine bacteria
IV.4.5 Horizontal gene transfer from marine to human gut bacteria
IV.4.6 Discussion
IV.4.7 Methods summary
IV.4.8 Methods
IV.4.9 References
IV.4.10 Supplementary material
IV.5 Introduction for manuscript3: Production of porphyran oligosaccharides with porphyranase
IV.6 Manuscript 3
IV.6.1 Abstract
IV.6.2 Introduction
IV.6.3 Material and Methods
IV.6.4 Results and discussion
IV.6.5 Conclusion
IV.6.6 References
V. Material and Methods
V.1 Expression and purification of PorA and PorB
V.2 DNA techniques and plasmid construction
V.3 The medium throughput cloning strategy
V.4 Screening for crystallization conditions
V.5 Kinetic studies
V.6 Sequences and phylogeny
V.7 Fluorophore-assisted carbohydrate electrophoresis analysis (PAGE)
V.8 Enzyme activity essays
VI. Final discussion and outlook
VI.1 The agarolytic system of Z. galactanivorans
VI.2 Screening for new marine glycoside hydrolases
VI.3 β-porphyranases discovery in marine bacteria
VI.4 Seaweed polysaccharide degrading CAZymes in human gut bacteria
VI.5 Marine glycoside hydrolases as tools to analyse marine POM and DOM.
VII. References
