Structural And Functional Studies On The Cellodextrin Phosphorylase TaCDP

Cellulose is the most abundant renewable biomass resource in nature,which can be used to replace fossil fuels and solve environmental problems.In addition,cellulose is also an important raw material for synthesis of environmentally friendly,biocompatible and high value biologic products.With the further study of microbial cellulose utilization model,cellulose disaccharide phosphorylase(CBP)and cellulose oligosaccharide phosphorylase(CDP)are the key enzymes in the efficient utilization of cellulose by microorganisms.They has been widely used in the metabolic pathway modification of Escherichia coli and yeast and other microorganisms.CDP from Thermosipho africanus(TaCDP)was characterized to have the catalytic ability of both CBP and CDP.The special ability will facilitate engineering of this enzyme to simplify the construction process of engineering bacteria for the co-utilization of cellulose disaccharide and cellulose oligosaccharide and strengthens the energy advantage of engineering bacteria.However,,the substrate extensiveness and bifunctional catalytic mechanism of TaCDP are still unclear for the low homology with the identified CBP and CDP.In this paper,the structure and function of TaCDP are studied by X-ray crystallography.The main results are as follows:(1)Recombinant TaCDP was overexpressed in Escherichia coli BL21(DE3)and purified to homogeneity by nickel column affinity chromatography,anion exchange chromatography and gel filtration chromatography.(2)The TaCDP crystal was screened through vapour diffusion method,and diffracted to 4.8 (?) resolution.Based on the anomalous diffraction data of Se-Met TaCDP crystal,the crystal structure of TaCDP was obtained.TaCDP is a modular protein,is a member of GH family 94 and is composed of four distinct domains:β-sandwich domain,helix linker,(α/α)6-barrel domain and curl domain.The catalytic amino acid residue D625 is located in the(α/α)6-barrel domain.(3)The crystal structure of mutant TaCDP(D625A)complex with substrate was obtained.inorganic phosphate,glycerol and cellobiose molecules binded in the active pocket of TaCDP.The overall structure information and the mutants activity analysis of TaCDP showed that R345,S891,S913,G914,H847 formed the binding sites of inorganic phosphoric acid,and R373,Y375,S891,R393,E840,D625 formed the binding sites of sugar.(4)Compared with homologous proteins,there are three long insertion sequences in the TaCDP sequence.The thermal stability of the deleted TaCDP mutants was 12～27℃ lower than that of the wild type(Tm=85℃),which indicated that these inserted sequences were essential for maintaining the stability of TaCDP.■In summary,Crystal structure determination of TaCDP and will provide critical insights into the structural basis of the broad substrate specificity of TaCDP.The complex structure reveals the composition of TaCDP substrate binding pocket.