Structures And Functions Of β-1,3-glucanase And β-1,3-glycosyltransferase

β-1,3-Glucan is a major natural polysaccharides and ubiquitous from all kingdoms of life, with many biological function. β-1,3-glucan metabolism enzymes system mainly contain three important enzymes:endo-β-1,3-glucanase (EC 3.2.1.39), exo-β-1,3-glucanase (EC 3.2.1.58) and β-1,3-glycosyltransferase (EC 2.4.1.-). Both of them catalyze β-1,3-glucan, form a series of catalytic products, and involve in the metabolizing of β-1,3-glucan in organism. In the present dissertation, structures, functions and applications of three β-1,3-glucan metabolism enzymes (GH family 64 β-1,3-glucanase, GH family 17β-1,3-glucanosyltransferase and GH family 16β-1,3-glucosyltransferase) were investigated. The main results are as follows:(1) A GH family 64β-1,3-glucanase gene (PbBg164A) from Paenibacillus barengoltzii was cloned and expressed in E. coli. The enzyme was a typical GH family 64 laminaripentaose-producing β-1,3-glucanase, could hydrolyze β-1,3-glucan to produce a series of oligosaccharides and laminaripentaose as the main product. Crystal structures of the free and laminarihexaose-bound forms of PbBg164A were resolved by X-ray crystallography. Based on sequence alignment and the activity determination, the domain A of.PbBg164A was identified as a new type of carbohydrate-binding motif, which exhibit a similar structure like insect β-1,3-glucan recognition protein (βGRP-N) Surprisingly, two sugar chains simultaneously interacted with one PbBg164A molecule in the complex structure. The spatial arrangement of the two sugar chains bound to PbBg164A was almost identical to that of the triple-helical β-1,3-glucan. On the basis of structural and functional analysis, we firstly reveal details of the triple-helical β-1,3-glucan recognition mode and hydrolytic mechanism of laminaripentaose produced by GH family 64 members. Perhaps more importantly, our results presented a potential structural basis on GH64-TLP-SF superfamily proteins (e.g. thaumatin-like proteins) about β-1,3-glucans recognition. Moreover, based on the utilization of PbBg164A hydrolyzing curdlan, we established methods for preparation, separation, purification and quantification of β-1,3-gluco-oligosaccharides with different polymerization degree.(2) A GH family 17 β-1,3-glucanosyltransferase gene (RmBgt17A) from Rhizomucor miehei was cloned and expressed in E. coli. The crystal structures of RmBgt17A and its two complexes were solved at resolutions of 1.30 A,2.30 A and 2.27 A, respectively. The overall structure of RmBgtl7A had the characteristic (β/α)8 TIM-barrel fold. The structures of RmBgt17A and other GH family 17 members were compared:it was found that a conserved subdomain, located in the terminal of the catalytic cleft in other GH family 17 members, was absent in RmBgt17A. Instead, four amino-acid residues exposed to the surface of the enzyme (Tyr135, Tyr136, Glu158 and His172) were found in the reducing terminal of subsite +2 of RwBgt17A, hindering access to the catalytic cleft. This distinct region of RmBgt17A makes its catalytic cleft shorter than that of other reported GH family 17 enzymes, which could only provide subsites-3 to+2. This structural evidence provides a clear explanation of RmBgt17A’s catalytic mode, whereby a laminaribiose is released from the reducing end of linear β-1,3-glucans and the remaining glucan is transferred to the end of another β-1,3-glucan as the acceptor to form a new β-1,6-linkage. A series of structural based site-directed mutagenesis studies indicated that a single Glu-to-Ala mutation (E158A) modulates the function of RmBgt17A to that of a endo-β-1,3-glucanase. Mutant E158A exhibited high hydrolytic activity (39.95 U/mg) toward reduced laminarin,348.5-fold higher than the wild type.(3) A novel fungal GH family 16 β-1,3-glucosyltransferase (PtBgt16A) from Paecilomyces thermophila was functionally and structurally characterized.PtBgt16A exhibited high transglycosylation activity towards laminari-oligosaccharides and cello-oligosaccharides, which transferred glucose residue to form high-polymeric oligosaccharides mixed β-1,3-and β-1,4-linkage. To investigate its substrate specificity and transglycosylation activities, the crystal structure of PtBgt16A has been determined by X-ray crystallography.PtBgt16A structure shows a unique ridgy loop in the middle of the catalytic groove. A key aromatic residue Tryl12 hinder the -2 site of the catalytic cleft groove, to form a catalytic pocket around the-1 site. This structural and functional evidence reveals that PtBgt16A exhibit an approximate exo-catalytic mode, whereby a glucose residue is cleaved from the non-reducing end of linear β-glucans and this is glucose residue then transferred to the non-reducing end of another β-glucan as the acceptor to form high-polymeric oligosaccharides.