| a-amylase are universally distributed throughout the animal, plant and microbial kingdoms. They can hydrolyse starch molecules to give diverse products including dextrins and progressively smaller polymers composed of glucose units, a-amylase are one of the most popular and important form of industrial amylases.The hydrolases and transferases in the a-amylase family are multidomain proteins which has a catalytic domain of a (α/β)8-barrel fold, with the active site at the C-terminal end of the β-strands. Although all the a-amylase enzymes contain similar catalytic domains and share a common functioning mechanism, they have been assigned to three separate glycoside hydrolase/transferase families GH13, GH70and GH77based on amino acid sequence similarity. Every a-amylase need one glutamic acid and two aspartic acid residues for its activity, while most enzymes of the family also contain two histidine residues critical for transition state stabilization. Those five residues occur in four short sequences conserved throughout the family, and within such sequences some key amino acid residues are related to enzyme specificity.AmyP is a raw starch-degrading a-amylase newly identified from a marine metagenome library. It shares low sequence similarity with characterized glycoside hydrolases and was classified into a new subfamily of GH13. Especially, it showed preferential degradation to raw rice starch. In the present study, full length AmyP was cloned and overexpressed in Escherichia coli, then purified and crystallized in the presence of its substrate analogue β-cyclodextrin. Single crystals were successfully obtained, and X-ray diffraction on one of the single crystals gained data to a resolution of2.1A. Preliminary analysis on the data indicated that AmyP might be a dimmer in the crystal, and the crystal belonged to space group P21212, with unit-cell parameters a=129.824A, b=215.534A, c=79.699A, α=90°,β=90°,γ=90°Only10%of amylases contain a distinct non-catalytic module that is known to facilitate binding and degradation of raw starch. Besides the common (α/β)8-barrel catalytic domain, these enzymes contain another β-barrel domain nominated starch binding domain (SBD) at the C-terminus, which is critical for the degradation of raw starch granules. SBDs belong to carbohydrate binding modules (CBMs), which have been classified into various CBM families. In the sequence-based classification, SBDs have been grouped into seven CBM families:CBM20, CBM21, CBM25, CBM26, CBM34, CBM41and CBM45. The SBDs in the family CBM20, including classical C-terminal SBDs of microbial amylases, are the most thoroughly studied SBDs. The three-dimensional structures of some SBDs have already been determined by X-ray crystallography or nuclear magnetic resonance. Similar to full length a-amylases, though the sequences of SBDs varies from each other, their three-dimensional structures are conserved. All SBDs adopt a β-barrel fold, with the activity center composed of a few conserved aromatic residues.To understand the functioning mechanism of AmyP especially its preferential activity to degrade raw starch granules, we further investigated the structure and function of the C-terminal putative starch binding domain of AmyP. In this study, AmyP-SBD was recombinantly expressed, purified, and characterized in structure and function by using circular dichroism (CD), nuclear magnetic resonance (NMR), and isothermal titration calorimetry (ITC). CD and NMR results indicated that AmyP-SBD adopted a molten globule like state. ITC and NMR data confirmed its substrate binding activity using P-cyclodextrin as substrate analogue. Intriguingly, NMR chemical shift perturbation experiments showed that AmyP-SBD experienced a conformational change from disorder to order when binding to P-cyclodextrin. This substrate binding coupled folding property was rarely observed on other SBDs. Further in depth study on the binding coupled folding of AmyP-SBD may be helpful to elucidate the functioning mechanism of AmyP and its application. |
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