Crystal Structures of <i>Escherichia coli</i> Branching Enzyme in Complex with Linear Oligosaccharides

Feng, Lei; Fawaz, Remie; Hovde, Stacy; Gilbert, Lindsey; Chiou, Janice; Geiger, James H.

doi:10.1021/acs.biochem.5b00228

Cited by 51 publications

(46 citation statements)

References 68 publications

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“…Among them, the C2 and C3 sites have not been described previously in any BE structure. In EcBE (PDB code 4LPC), oligosaccharide binding was found at SBSs corresponding to the 48_1, A1, A2, and C1 sites (24). In OsBEI (PDB code 3VU2), sugar chains were bound at SBSs corresponding to the 48_1 and 48_2 sites (23).…”

Section: Resultsmentioning

confidence: 99%

“…van der Waals contacts were also involved in the binding of the sugar ( Table 2). The A1 site corresponds to sugar-binding site III described in EcBE (24), in which the maltose (G2) portion of G7 was bound. However, the important Tyr 500 and Asp 501 residues in cceBE1 were replaced by Asp 471 and Met 472 , respectively, in EcBE.…”

Section: Resultsmentioning

confidence: 99%

“…4D and Table 2). Sugar-binding site VI described in EcBE (24), corresponding to the A2 site, had the G3 portion of G7, and important amino acids for recognition of the maltooligosaccharide were conserved (Fig. 4D).…”

Section: Table 2 Interactions Between Bound Sugars and Amino Acid Resmentioning

confidence: 99%

“…These studies provided basic information on the overall and domain structures of GH13_9 (prokaryotic)-and GH13_8 (eukaryotic)-type BEs. The structures of OsBEI (23), HsBE (22), and EcBE (24,25) in complex with linear maltooligosaccharides or cyclodextrins led to the identification of surface binding sites (SBSs) located on the enzyme surface at certain distances from the active site (26 -29). Possible roles of SBSs (substrate targeting, guiding substrate into the active site, and passing on reactions products) have been proposed (27).…”

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confidence: 99%

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Bound Substrate in the Structure of Cyanobacterial Branching Enzyme Supports a New Mechanistic Model

Hayashi

Suzuki

Colleoni

et al. 2017

Journal of Biological Chemistry

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Branching enzyme (BE) catalyzes the formation of α-1,6-glucosidic linkages in amylopectin and glycogen. The reaction products are variable, depending on the organism sources, and the mechanistic basis for these different outcomes is unclear. Although most cyanobacteria have only one BE isoform belonging to glycoside hydrolase family 13, sp. ATCC 51142 has three isoforms (BE1, BE2, and BE3) with distinct enzymatic properties, suggesting that investigations of these enzymes might provide unique insights into this system. Here, we report the crystal structure of ligand-free wild-type BE1 (residues 5-759 of 1-773) at 1.85 Å resolution. The enzyme consists of four domains, including domain N, carbohydrate-binding module family 48 (CBM48), domain A containing the catalytic site, and domain C. The central domain A displays a (β/α)-barrel fold, whereas the other domains adopt β-sandwich folds. Domain N was found in a new location at the back of the protein, forming hydrogen bonds and hydrophobic interactions with CBM48 and domain A. Site-directed mutational analysis identified a mutant (W610N) that bound maltoheptaose with sufficient affinity to enable structure determination at 2.30 Å resolution. In this structure, maltoheptaose was bound in the active site cleft, allowing us to assign subsites -7 to -1. Moreover, seven oligosaccharide-binding sites were identified on the protein surface, and we postulated that two of these in domain A served as the entrance and exit of the donor/acceptor glucan chains, respectively. Based on these structures, we propose a substrate binding model explaining the mechanism of glycosylation/deglycosylation reactions catalyzed by BE.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Table 2 Interactions Between Bound Sugars and Amino Acid Resmentioning

confidence: 99%

mentioning

confidence: 99%

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Bound Substrate in the Structure of Cyanobacterial Branching Enzyme Supports a New Mechanistic Model

Hayashi

Suzuki

Colleoni

et al. 2017

Journal of Biological Chemistry

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“…OsttaBE structure was modeled using starch debranching enzyme from Hordeum vulgare (PDB code 4J3S)[54]. Oryza sativa and E. coli BE crystal structure´s (PDB codes 3AMK and 5E6Z, respectively) were used to compare catalytic domains[55][56][57]. Alignment with the template was based on homology and secondary structure.…”

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confidence: 99%

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

Hedín

Barchiesi

Gómez-Casati

et al. 2017

Archives of Biochemistry and Biophysics

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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.

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Site‐directed mutagenesis of Serine‐72 reveals the location of the fructose 6‐phosphate regulatory site of the Agrobacterium tumefaciensADP‐glucose pyrophosphorylase

et al. 2022

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The allosteric regulation of ADP-glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition.

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Crystal Structures of Escherichia coli Branching Enzyme in Complex with Linear Oligosaccharides

Cited by 51 publications

References 68 publications

Bound Substrate in the Structure of Cyanobacterial Branching Enzyme Supports a New Mechanistic Model

Bound Substrate in the Structure of Cyanobacterial Branching Enzyme Supports a New Mechanistic Model

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

Site‐directed mutagenesis of Serine‐72 reveals the location of the fructose 6‐phosphate regulatory site of the Agrobacterium tumefaciensADP‐glucose pyrophosphorylase

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