Identification and characterization of a novel starch branching enzyme from the picoalgae Ostreococcus tauri

Abstract

Starch branching enzyme is a highly conserved protein from plants to algae. This enzyme participates in starch granule assembly by the addition of α-1,6-glucan branches to the α-1,4-polyglucans. This modification determines the structure of amylopectin thus arranging the final composition of the starch granule. Herein, we describe the function of the Ot01g03030 gene from the picoalgae Ostreococcus tauri. Although in silico analysis suggested that this gene codes for a starch debranching enzyme, our biochemical studies support that this gene encodes a branching enzyme (BE). The resulting 1058 amino acids protein has two in tandem carbohydrate binding domains (CBMs, from the CBM41 and CBM48 families) at the N-terminal (residues 64-403) followed by the C-terminal catalytic domain (residues 426-1058). Analysis of the BE truncated isoforms show that the CBMs bind differentially to whole starch, amylose or amylopectin. Furthermore, both CBMs seem to be essential for BE activity, as no catalytic activity was detected in the truncated enzyme comprising only by the catalytic domain. Our results suggest that the Ot01g03030 gene codifies for a functional BE containing two CBMs from CBM41 and CBM48 families which are critical for enzyme function and regulation.

Starch Branching Enzyme 1 Is Important for Amylopectin Synthesis and Cyst Reactivation in Toxoplasma gondii

“…Recombinant protein SBE1 enzymatic activity assay was detected according to previous research ( 32 , 33 ). Briefly, 10 μL recombinant protein was incubated with different concentrations of amylose (10 to 1,500 μg/mL) in 180 μL 50 mM NaCl in PBS buffer (pH = 7.0) at 37°C for 30 min.…”

Bacterial transcriptional response to labile exometabolites from photosynthetic picoeukaryote Micromonas commoda

“…These two enzymes are suggestive of the presence of a α-1,4-glucan in the exometabolome of Micromonas . Members of the green algal lineage, like their true plant relatives, carry genes for carbon storage as starch, consisting of α-1,4-glucan units as amylose and amylopectin [ 58 , 59 ]. In PUL5, enriched genes included an N -acetylglucosamine kinase.…”

Functional and Structural Characterization of a Novel Isoamylase from Ostreococcus tauri and Role of the N-Terminal Domain

“…Other aminoacids that are well conserved are Glu399 (Glu526 in OsttaISA1) and Asp471 (Asp597 on OsttaISA1) Table 1. Numerous studies analyzing the role of these amino acids in the GH13 proteins showed that Asp363 acts as a catalytic nucleophile and would form a covalent intermediate by binding to the glucose moiety at the reducing end of the molecule, while Glu399 is the catalytic acid/base that would protonate the oxygen from the -1 glucose [29,51,58]. These aminoacids are involved in the so-called double displacement mechanism, which is proposed for all the reactions involving GH13 enzymes [59].…”

“…Fig. (3A) shows in magenta the position of this putative CBM from OsttaISA1, and it can be clearly observed that it comprises six ß-strands forming the classical CBM ß-sandwich folding [27,29]. The central part of the protein is composed of the Catalytic Domain (CD), which is also conserved among the α-amylase family, and belongs to the family 13 of glycoside hydrolase from the CAZY database [51,61].…”

“…On the other hand, the lower affinity for amylose seems to be related to the linear structure of the molecule. The behavior with starch might respond to a steric hindrance, as seems to be the case of CBM41 from the branching enzyme from Ostreococcus tauri [29].…”