The Crystal Structure of Nitrosomonas Europaea Sucrose Synthase Reveals Critical Conformational Changes and Insights into the Sucrose Metabolism in Prokaryotes

In this paper we report the first crystal structure of a prokaryotic sucrose synthase from the non-photosynthetic bacterium Nitrosomonas europaea. The obtained structure was in an open form, whereas the only other available structure from the plant Arabidopsis thaliana was in a closed conformation. Comparative structural analysis revealed a “hinge-latch” combination, which is critical to transition between the open and closed forms of the enzyme. The N. europaea sucrose synthase shares the same fold as the GT-B family of the retaining glycosyltransferases. In addition, a triad of conserved homologous catalytic residues in the family showed to be functionally critical in the N. europaea sucrose synthase (Arg567, Lys572, Glu663). This implies that sucrose synthase shares not only a common origin with the GT-B family, but also a similar catalytic mechanism. The enzyme preferred transferring glucose from ADP-glucose rather than UDP-glucose like the eukaryotic counterparts. This predicts that these prokaryotic organisms have a different sucrose metabolic scenario from plants. Nucleotide preference determines where the glucose moiety is targeted after sucrose is degraded

Structural analysis reveals a pyruvate-binding activator site in the Agrobacterium tumefaciens ADP–glucose pyrophosphorylase

The pathways for biosynthesis of glycogen inbacteria and starch in plants are evolutionarily andbiochemically related. They are regulated primarily by ADP?glucose pyrophosphorylase, which evolved to satisfy metabolic requirements of a particular organism. Despite the importance of these two pathways, little is known about the mechanism that controls pyrophosphorylase activity or the location of its allosteric sites. Here, we report pyruvate-bound crystal structures of ADP-glucose pyrophosphorylase from the bacterium Agrobacterium tumefaciens, identifying a previously elusive activator site for the enzyme. We found that the tetrameric enzyme binds two molecules of pyruvate in a planar conformation. Each binding site is located in a crevice between the C-terminal domains of two subunits where they stack via a distinct β-helix region. Pyruvate interacts with the side chain of Lys-43 and with the peptide backbone of Ser-328 and Gly-329 from both subunits. These structural insights led to the design of two variants with altered regulator properties. In one variant (K43A), the allosteric effect was absent, whereas in the other (G329D), the introduced Asp mimicked the presence of pyruvate. The latter generated an enzyme that was pre-activated and insensitive to further activation by pyruvate. Our study furnishes a deeper understanding of how glycogen biosynthesis is regulated in bacteria and the mechanism by which transgenic plants increased their starch production. These insights will facilitate rational approaches to enzyme engineering for starch production in crops of agricultural interest and will promote further study of allosteric signal transmission and molecular evolution in this important enzyme family.Fil: Hill, B. L.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados UnidosFil: Mascarenhas, R.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados UnidosFil: Patel, H. P.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados UnidosFil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Wu, R.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados UnidosFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Liu, D.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados UnidosFil: Ballicora, M. A.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados Unido

A novel dual allosteric activation mechanism of Escherichia coli ADP-glucose pyrophosphorylase: the role of pyruvate.

Fructose-1,6-bisphosphate activates ADP-glucose pyrophosphorylase and the synthesis of glycogen in Escherichia coli. Here, we show that although pyruvate is a weak activator by itself, it synergically enhances the fructose-1,6-bisphosphate activation. They increase the enzyme affinity for each other, and the combination increases Vmax, substrate apparent affinity, and decreases AMP inhibition. Our results indicate that there are two distinct interacting allosteric sites for activation. Hence, pyruvate modulates E. coli glycogen metabolism by orchestrating a functional network of allosteric regulators. We postulate that this novel dual activator mechanism increases the evolvability of ADP-glucose pyrophosphorylase and its related metabolic control

A Novel Dual Allosteric Activation Mechanism of <i>Escherichia coli - Figure 3 </i> ADP-Glucose Pyrophosphorylase: The Role of Pyruvate

<p>A) Effect of Pyr on the saturation curves for Fru-1,6-P₂. Assays were performed in the absence (control, ○), or the presence of Pyr (2.5 mM, •; or 10 mM, ▴). B) Effect of Pyr on the saturation curves for PLP. Assays were performed in the absence (control, ○), or the presence of 10 mM Pyr (▴). Other conditions for the assays were as described under Materials and Methods.</p

<i>E. coli</i> ADP-Glc PPase Pyr saturation curves in the absence (control, ○) or presence of Fru-1,6-P₂ (10 µM, ▪; or 30 µM, ▾).

<p>Other conditions for the assays were as described under Materials and Methods. •□▴.</p

Effect of Pyr on the AMP inhibition.

<p>Assays were performed in the absence (control, ○), or the presence of 20 mM Pyr (•). Other conditions for the assays were as described under Materials and Methods. Activity is relative to experiments conducted in absence or presence of Pyr, whose absolute values were 31.2 and 61.8 U/mg, respectively.</p

Effect of Fru-1,6-P₂ on the apparent affinity of ADP-Glc PPase for Pyr.

<p>Other conditions for the assays were as described under Materials and Methods. Values are the mean of three independent measurements ± standard deviation.</p

Proposed dual allosteric model for <i>E. coli</i> ADP-Glc PPase.

<p>The two main domains of the enzyme are illustrated by C and N. Catalysis occurs in the N-domain where the active site for ATP and Glc-1P is located. Full and dashed arrows indicate a strong and weak positive interaction, respectively.</p

Kinetic parameters for <i>E. coli</i> ADP-Glc PPase.

<p>Assays were carried out as described in Material and Methods. Values are average numbers from three independent experiments, using regression analysis.</p