Abstract
Background: The debranching starch enzymes, isoamylase 1 and 2 are well-conserved enzymes present in almost all the photosynthetic organisms. These enzymes are involved in the crystallization process of starch and are key components which remove misplaced α-1,6 ramifications on the final molecule. Aim: In this work, we performed a functional and structural study of a novel isoamylase from Ostreococcus tauri. Methods: We identified conserved amino acid residues possibly involved in catalysis. We also identified a region at the N-terminal end that resembles a Carbohydrate Binding Domain (CBM), which is more related to the family CBM48, but has no spatial conservation of the residues involved in carbohydrate binding. Results: The cloning, expression and biochemical characterization of this N-terminal region confirmed that it binds to polysaccharides, showing greater capacity for binding to amylopectin rather than total starch or amylose. Conclusion: This module could be a variant of the CBM48 family or it could be classified within a new CBM family.
Highlights
Carbohydrates are the most abundant molecules on earth; each year photosynthesis fixes approximately 100 billion tons of CO2 and H2O into cellulose, starch, sucrose and other sugars
The cloning, expression and biochemical characterization of this N-terminal region confirmed that it binds to polysaccharides, showing greater capacity for binding to amylopectin rather than total starch or amylose
This module could be a variant of the carbohydrate-binding domain from family 48 (CBM48) family or it could be classified within a new Carbohydrate Binding Domain (CBM) family
Summary
Carbohydrates are the most abundant molecules on earth; each year photosynthesis fixes approximately 100 billion tons of CO2 and H2O into cellulose, starch, sucrose and other sugars. CBM can be composed of 30 to 200 amino acids and can be as a sole domain or as a part of a tandem According to their sequence, structure and substrate binding specificity, CBM can be classified into 86 families [15]. Structure and substrate binding specificity, CBM can be classified into 86 families [15] They can serve different purposes, and the reason why they are present on the proteins may vary. SBDs are found in archaea, bacteria and eukaryotes and are distributed along twelve families of CBM: 20, 21, 25, 26, 34, 41, 45, 48, 53, 58, 68, & 69 (http://www.cazy.org/) These SBD modules have the evolutionary advantage of being able to disrupt the surface of the substrate due to the presence of two different binding sites [16 - 18]. These enzymes are involved in the crystallization process of starch and are key components which remove misplaced α-1,6 ramifications on the final molecule
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