A Bacterial Glucanotransferase Can Replace the Complex Maltose Metabolism Required for Starch to Sucrose Conversion in Leaves at Night

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Crystal Structures of the Catalytic Domain of Arabidopsis thaliana Starch Synthase IV, of Granule Bound Starch Synthase From CLg1 and of Granule Bound Starch Synthase I of Cyanophora paradoxa Illustrate Substrate Recognition in Starch Synthases

Starch synthases (SSs) are responsible for depositing the majority of glucoses in starch. Structural knowledge on these enzymes that is available from the crystal structures of rice granule bound starch synthase (GBSS) and barley SSI provides incomplete information on substrate binding and active site architecture. Here we report the crystal structures of the catalytic domains of SSIV from Arabidopsis thaliana, of GBSS from the cyanobacterium CLg1 and GBSSI from the glaucophyte Cyanophora paradoxa, with all three bound to ADP and the inhibitor acarbose. The SSIV structure illustrates in detail the modes of binding for both donor and acceptor in a plant SS. CLg1GBSS contains, in the same crystal structure, examples of molecules with and without bound acceptor, which illustrates the conformational changes induced upon acceptor binding that presumably precede catalytic activity. With structures available from several isoforms of plant and non-plant SSs, as well as the closely related bacterial glycogen synthases, we analyze, at the structural level, the common elements that define a SS, the elements that are necessary for substrate binding and singularities of the GBSS family that could underlie its processivity. While the phylogeny of the SSIII/IV/V has been recently discussed, we now further report the detailed evolutionary history of the GBSS/SSI/SSII type of SSs enlightening the origin of the GBSS enzymes used in our structural analysis

Model describing the effect of the L78 insertion on polysaccharide binding to <i>Hv</i>Pho1.

<p>(A) <i>Hv</i>Pho1 forms a homodimer in solution with an enlarged molecular size. The formation of the dimer is brought about by the crystallographic dimer interface. The flexible nature of the L78 insertion could block access of larger glucans to the protein´s surface. (B) The specific degradation products of <i>Hv</i>Pho1 are the F50s which probably lack L78. Our crystal structure does not contain the L78 insertion and might therefore represent the F50s rather than the full-length enzyme. The F50s provide better access to larger polysaccharides like starch or amylopectin. (C) <i>Hv</i>Pho1ΔL78 lacks the L78 insertion but it also lacks a break in the protein chain. Affinity of larger polysaccharides is similar to full-length <i>Hv</i>Pho1 as the main protein backbone is closed and restricts access to this area.</p

Functional and structural characterization of plastidic starch phosphorylase during barley endosperm development

<div><p>The production of starch is essential for human nutrition and represents a major metabolic flux in the biosphere. The biosynthesis of starch in storage organs like barley endosperm operates via two main pathways using different substrates: starch synthases use ADP-glucose to produce amylose and amylopectin, the two major components of starch, whereas starch phosphorylase (Pho1) uses glucose-1-phosphate (G1P), a precursor for ADP-glucose production, to produce α-1,4 glucans. The significance of the Pho1 pathway in starch biosynthesis has remained unclear. To elucidate the importance of barley Pho1 (<i>Hv</i>Pho1) for starch biosynthesis in barley endosperm, we analyzed <i>Hv</i>Pho1 protein production and enzyme activity levels throughout barley endosperm development and characterized structure-function relationships of <i>Hv</i>Pho1. The molecular mechanisms underlying the initiation of starch granule biosynthesis, that is, the enzymes and substrates involved in the initial transition from simple sugars to polysaccharides, remain unclear. We found that <i>Hv</i>Pho1 is present as an active protein at the onset of barley endosperm development. Notably, purified recombinant protein can catalyze the <i>de novo</i> production of α-1,4-glucans using <i>Hv</i>Pho1 from G1P as the sole substrate. The structural properties of <i>Hv</i>Pho1 provide insights into the low affinity of <i>Hv</i>Pho1 for large polysaccharides like starch or amylopectin. Our results suggest that <i>Hv</i>Pho1 may play a role during the initiation of starch biosynthesis in barley.</p></div

Structural details of the active site of <i>Hv</i>Pho1 and acceptor recognition.

<p>(A) Mode of binding of maltotetraose in the active site of <i>Hv</i>Pho1 (which is depicted as a semi-transparent ribbon). Maltotetraose is shown as ball and stick with yellow carbons and can also be seen in the same color in panel C. A modeled maltose is in ball and stick with orange carbons. Contacting amino acids are shown as stick models with green carbons, while those involved in stacking interactions with the glucose units are depicted with gray carbons. The pyridoxal phosphate is also depicted, with pink carbons, in the lower left corner. (B) Movement of a loop of <i>Hv</i>Pho1 in response to maltotetraose binding. Residues 422–427 of the <i>Hv</i>Pho1 complex with maltotetraose are highlighted with all atom sticks (orange carbons) and the maltotetraose is shown as sticks with cyan carbons. The thicker ribbons represent the α-carbon trace of <i>Hv</i>Pho1 bound to maltotetraose (orange), <i>Hv</i>Pho1 in the native structure (green), <i>Hv</i>Pho1 in complex with acarbose (gray), <i>Ec</i>MalP in complex with maltopentaose (yellow) and rabbit muscle glycogen phosphorylase (white). (C) Superposition of maltopentaose in the binding site of MalP (white, from PDB_code 1e4o), maltotetraose in <i>Hv</i>Pho1 and acarbose in <i>Hv</i>Pho1; plus PLP groups and selected details from the native <i>Hv</i>Pho1 structure. For clarity, only the mentioned groups, the pyridoxal-5’-phosphates (PLP) and, in the case of all three structures of <i>Hv</i>Pho1 reported here, Tyr900 and Tyr905 are depicted. Details from the maltotetraose complex are shown with yellow carbons, details from the acarbose complex with cyan carbons and details from the native structure with pink carbons. The four glucose units in the maltotetraose complex overlap well with the MalP structure for sub-sites +1 to +4.</p

Apparent molecular weights and affinity of <i>Hv</i>Pho1 constructs.

<p>(A), (B), (C) Total barley endosperm soluble protein loaded onto a 26/60 Superdex S-200 SEC column. (A) Protein fractions as analyzed by SDS-PAGE/immunoblot using anti-<i>Hv</i>Pho1 polyclonal antibodies and (B) Native gels using glycogen as in gel glycosyl acceptor and G1P as glycosyl donor. The first lane (marked M) on each immunoblot is protein molecular weight ladder. The first lane in each native gel (marked C) is the recombinant <i>Hv</i>Pho1 control. Arrows on top of the gels indicate molecular weight of the protein fraction according to column calibration. (C) Chemical cross-linking of <i>Hv</i>Pho1 dimers in solution. <i>Hv</i>Pho1 was incubated for the indicated times with 0,15% (v/v) glutaraldehyde. Formation of <i>Hv</i>Pho1 cross-linked dimers is indicated. (D) Protein fractions as analyzed by SDS-PAGE/immunoblot using anti-<i>Hv</i>BeIIb polyclonal antibodies. Total barley endosperm protein was either incubated either with (left gel) or without (right gel) 1 mM ATP and 2.5 mM protein phosphatase inhibitor cocktail prior to size separation via SEC. Lane 1: protein molecular weight ladder, lanes marked 2: protein fractions from SEC ranging between 330 kDa and 190 kDa; lane 3: recombinant <i>Hv</i>BeIIb purified from <i>E</i>. <i>coli</i>. Brown arrows to the right of both gels indicate HvBeIIb. (E) SEC profile of <i>Hv</i>Pho1 (blue) and <i>Hv</i>Pho1ΔL78 (black). The inserts are DLS analyses of the hydrodynamic sizes of the two proteins. The green and brown lines represent the SEC peak fractions of the respective proteins used for SEC analysis. Arrows on top indicate molecular weight according to column calibration. (F) The affinity of recombinant <i>Hv</i>Pho1, <i>Hv</i>Pho1F50 and <i>Hv</i>Pho1ΔL78 for amylopectin and starch assessed by analysis of starch bound and unbound protein <i>in vitro</i> in two ways: Top: By incubation of the proteins with 5, 1 and 0.5 mg∙ml^-1 amylopectin and successive analysis of soluble (S) and pellet–amylopectin bound (P) fraction with SDS-PAGE. The concentrations of amylopectin are indicated over each S/P pair. Bottom: analysis via native gel with in gel starch as an interaction partner. Rf values are plotted versus the starch concentration in the gels. The resultant affinity constants for half maximum binding are given.</p

Abundance levels and enzymatic activity of <i>Hv</i>Pho1 during barley endosperm development.

<p>(A) <i>Hv</i>Pho1 protein abundance from barley endosperm extracts analyzed via immunoblot at 2 d intervals. Only one band is visible just above 100 kDa in accordance with an expected mass of 105 kDa. (B) Relative quantification of the data from panel A (blue line) and activity from panel C (H₂O control; red line). (C) Starch phosphorylase activity probed in 2 day intervals as for panel A but with native gels and Lugol coloring of activity products. Strong synthetic activity appears as a dark stained band and is marked with a black arrow. White bands and smears represent amylolytic activities. All gels include a recombinant <i>Hv</i>Pho1 control as the right-most band. The different redox treatments are indicated next to each gel. (D) Immunoblot (top) and native gel (bottom) analysis of <i>Hv</i>Pho1 protein abundance on buffer soluble (S) protein and buffer insoluble (P) protein fractions of barley endosperm between 0 and 8 DAF. Numbers indicate the DAF. Arrows mark the position of the two relevant bands in the immunoblot and the position of the (single) activity band in the zymogram.</p